SKI-606

Pharmacological Research xxx (2015) xxx–xxx

1 Invited Review
2 Src protein-tyrosine kinase structure, mechanism, and small molecule
3 inhibitors
4 Q1 Robert Roskoski Jr. ∗

5 Blue Ridge Institute for Medical Research, 3754 Brevard Road, Suite 116, Box 19, Horse Shoe, NC 28742-8814, United States
6

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a r t i c l e i n f o

a b s t r a c t

9 Article history:
10 Received 26 January 2015
11 Accepted 26 January 2015
12 Available online xxx

13 This paper is dedicated to the memory of
14 Prof. Donald F. Steiner (1930–2014) –
15 advisor, mentor, and discoverer of
16 proinsulin.
17
18 Chemical compounds studied in this article:
19 Bosutinib (PubMed CID: 5328940)
20 Dasatinib (PubMed CID: 3062316)
21 Ponatinib (PubMed CID: 24826799)
22 Saracatinib (PubMed CID: 10302451)
23 Vandetanib (PubMed CID: 3062316)

24 Keywords:
25 BCR-Abl
26 Catalytic spine
27 Regulatory spine
28 SH2 domain
29 SH3 domain
30 Targeted cancer therapy

The physiological Src proto-oncogene is a protein-tyrosine kinase that plays key roles in cell growth, division, migration, and survival signaling pathways. From the N- to C-terminus, Src contains a unique domain, an SH3 domain, an SH2 domain, a protein-tyrosine kinase domain, and a regulatory tail. The chief phosphorylation sites of human Src include an activating pTyr419 that results from phosphory- lation in the kinase domain by an adjacent Src molecule and an inhibitory pTyr530 in the regulatory tail that results from phosphorylation by C-terminal Src kinase (Csk) or Chk (Csk homologous kinase). The oncogenic Rous sarcoma viral protein lacks the equivalent of Tyr530 and is constitutively activated. Inactive Src is stabilized by SH2 and SH3 domains on the rear of the kinase domain where they form an immobilizing and inhibitory clamp. Protein kinases including Src contain hydrophobic regulatory and catalytic spines and collateral shell residues that are required to assemble the active enzyme. In the inactive enzyme, the regulatory spine contains a kink or a discontinuity with a structure that is incom- patible with catalysis. The conversion of inactive to active Src is accompanied by electrostatic exchanges involving the breaking and making of distinct sets of kinase domain salt bridges and hydrogen bonds. Src-catalyzed protein phosphorylation requires the participation of two Mg2+ ions. Although nearly all protein kinases possess a common K/E/D/D signature, each enzyme exhibits its unique variations of the protein-kinase reaction template. Bosutinib, dasatinib, and ponatinib are Src/multikinase inhibitors that are approved by the FDA for the treatment of chronic myelogenous leukemia and vandetanib is approved for the treatment of medullary thyroid cancer. The Src and BCR-Abl inhibitors saracatinib and AZD0424, along with the previous four drugs, are in clinical trials for a variety of solid tumors including breast and lung cancers. Both ATP and targeted therapeutic Src protein kinase inhibitors such as dasatinib and ponatinib make hydrophobic contacts with catalytic spine residues and form hydrogen bonds with hinge residues connecting the small and large kinase lobes.

32 Contents

33 Introduction 00
34 Organization of Src 00
35 SH3, SH2, SH1 domains 00
36 Secondary structure of the Src protein kinase domain: the protein kinase fold 00
37 Interconversion of the autoinhibited and active conformations of Src 00
38 Src regulation by the latch, clamp, and switch 00
39 Unlatching by phosphatases 00

Abbreviations: AL, activation loop; ALL, acute lymphoblastic leukemia; AS, activation segment; CDK, cyclin-dependent kinase; Chk, Csk homologous kinase; CML, chronic myelogenous leukemia; Csk, C-terminal Src kinase; C-spine, catalytic spine; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated protein kinase; FGFR, ﬁbroblast growth factor receptor; GIST, gastrointestinal stromal tumor; HGFR or c-Met, hepatic growth factor receptor; H8, hydrophobic; IGFR, insulin-like growth factor receptor; NSCLC, non-small cell lung cancer; PDGFR, platelet-derived growth factor receptor; Ph+, Philadelphia chromosome positive; PKA, protein kinase A; PTP, protein-tyrosine phosphatase; pTyr or pY, phosphotyrosine; R-spine, regulatory spine; Sh, shell; SH1/2/3, Src homology 1/2/3; VEGFR, vascular endothelial growth factor receptor.
∗ Tel.: +1 828 891 5637; fax: +1 828 890 8130.
E-mail address: [email protected]

http://dx.doi.org/10.1016/j.phrs.2015.01.003

2 R. Roskoski Jr. / Pharmacological Research xxx (2015) xxx–xxx

Switching and unclamping 00
Conversion of active to inactive Src 00
Structure of the Src kinase domain skeleton 00
The regulatory spine 00
The catalytic spine 00
Spinal collateral ligaments or shell residues 00
Src catalytic residues 00
Properties of the small and large lobes 00
The K/E/D/D protein kinase signature 00
Stabilizing the Src activation segment 00
Role of magnesium ions in the protein kinase catalytic process 00
Participation of two magnesium ions in catalysis 00
Targeting the Mg2+binding sites 00
Src signaling and cancer 00
Therapeutic small molecule Src inhibitors 00
Src as a drug target 00
Src inhibitors that are FDA-approved or in clinical trials 00
The ATP-binding pocket of Src 00
ATP-competitive Src inhibitors 00
Epilogue 00
Conﬂict of interest 00
Acknowledgement 00
Appendix A. Supplementary data 00
References 00

40 Introduction
Q2
41 Src, a non-receptor protein-tyrosine kinase, has been the sub-
42 ject of intense investigation for three decades owing in part to its
43 association with malignant transformation and oncogenesis. These
44 studies stem from work on the Rous sarcoma virus, a chicken tumor
45 virus discovered in 1911 by Peyton Rous [1]. v-Src (a viral protein)
46 is encoded by the avian cancer-causing oncogene of Rous sarcoma
47 virus. In contrast, Src (the normal cellular homologue) is encoded
48 by a physiological gene, the ﬁrst proto-oncogene to be described
49 and characterized [2].
50 Src is expressed ubiquitously in vertebrate cells; however, brain,
51 osteoclasts, and platelets express 5–200 fold higher levels of this
52 protein than most other cells [3]. In ﬁbroblasts, Src is bound to
53 endosomes, perinuclear membranes, secretory vesicles, and the
54 cytoplasmic face of the plasma membrane where it can interact
55 with a variety of growth factor, integrin, and G-protein-coupled
56 receptors and serve as an essential intermediary in signal transduc-
57 tion [3,4]. The expression of high levels of Src in platelets (anucleate
58 cells) and in neurons (which are postmitotic) indicates that Src
59 participates in processes other than cell division [3].
60 Protein kinases including Src catalyze the following reaction:
61 MgATP1− + protein–O : H → protein–O : PO32− + MgADP + H+
62 Note that the phosphoryl group (PO32–) and not the phosphate

Protein phosphorylation is the most widespread class of post- translational modiﬁcation used in signal transduction [5]. Families of protein phosphatases catalyze the dephosphorylation of pro- teins thus making phosphorylation-dephosphorylation an overall reversible process [7]. Protein kinases play a predominant regula- tory role in nearly every aspect of cell biology [5]. They regulate apoptosis, cell cycle progression, cytoskeletal rearrangement, dif- ferentiation, development, the immune response, nervous system function, and transcription. Src and the Src family kinases have been implicated in each of these processes [3,4]. Moreover, dysregula- tion of protein kinases occurs in a variety of diseases including cancer and inﬂammatory disorders. Considerable effort has been expended to determine the manifold functions of protein kinase signal transduction pathways during the past 50 years.
Manning et al. included 11 members in the human Src kinase
family [5]. The four closely related group I enzymes include Src, Fyn, Yes, and Fgr and the four closely related group II enzymes include Blk, Hck, Lck, and Lyn. The three group III enzymes, which are dis- tantly related to these two groups, include Frk, Srm, and Brk. Src, Fyn, and Yes are expressed in all cell types [3]. In contrast, Blk, Fgr, Hck, Lck, and Lyn are found primarily in hematopoietic cells, and Srm is found in keratinocytes. Frk occurs chieﬂy in bladder, brain, breast, colon, and lymphoid cells. Moreover, Brk occurs chieﬂy in colon, prostate, and small intestine; however, it was initially iso- lated from a breast cancer cell line [8].

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63 (OPO32–) group is transferred from ATP to the protein substrate.
64 Divalent cations such as Mg2+ are required for the reaction. Based Organization of Src 103
65 upon the nature of the phosphorylated OH group, these enzymes
66 are classiﬁed as protein-tyrosine, protein-serine/threonine, or dual SH3, SH2, SH1 domains 104
67 speciﬁcity protein-tyrosine/threonine kinases.
68 Manning et a. identiﬁed 478 typical and 40 atypical protein From the N- to C-terminus, Src contains an N-terminal 14- 105
69 kinase genes in humans (total 518) [5]. The family includes 90 carbon myristoyl group, a unique domain, an SH3 domain, an SH2 106
70 protein-tyrosine kinases, 43 tyrosine-kinase like proteins, and domain, an SH2-kinase linker, a protein-tyrosine kinase domain 107
71 385 protein-serine/threonine kinases. Of the 90 protein-tyrosine (SH1), and a C-terminal regulatory segment (Fig. 1) [3,4]. During 108
72 kinases, 58 are receptor and 32 are non-receptor enzymes including biosynthesis, the amino-terminal methionine is removed and the 109
73 Src. A small group of dual-speciﬁcity kinases including MEK1 and resulting amino-terminal glycine becomes myristoylated following 110
74 MEK2 catalyze the phosphorylation of both tyrosine and threonine its reaction with myristoyl-CoA. 111
75 in target proteins; dual-speciﬁcity kinases possess molecular fea- The human SRC gene encodes 536 amino acids and the chicken 112
76 tures that place them within the protein-serine/threonine kinase Src gene encodes 533 residues while the avian Rous sarcoma viral 113
77 family [6]. Src oncogene encodes 526 residues. The human and chicken Src 114

R. Roskoski Jr. / Pharmacological Research xxx (2015) xxx–xxx 3

Fig. 1. Organization of human Src. CL, catalytic loop; AS, activation segment.

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proteins Exhibit 99.6% identity with most of the variation occurring near the N-terminus. The chicken viral Src (v-Src) protein Exhibits 95.8% identity with chicken Src. The protein kinase domains of the chicken/human proto-oncogenes (residues 267/270–520/523) exhibit only two differences: chicken Met354 corresponds to human Thr357 and chicken Asp502 corresponds to human Glu505. Thus, results from studies of the protein kinase domain of chicken Src are expected to accurately reﬂect those of the human enzyme. The C-terminal tails (residues 521/524–533/536) of chicken/human Src are identical, but they are completely dif- ferent from those of Rous v-Src. The Rous viral oncogene protein (v-Src) lacks seven-residues at its carboxyterminus, which include an autoinhibitory tyrosine phosphorylation site, thus accounting for its increased basal activity. Human Src and the Rous v-Src protein kinase domains Exhibit 11 residue differences. The chicken numbering system is used in much of the early literature, even when studies were performed with the human enzyme. In this paper, unless speciﬁed otherwise, human residue numbers are used for both human and chicken Src reﬂecting efforts targeting the human enzyme for drug discovery. Three amino acids in the avian protein are deleted after human Src residue 25. To go from
the human Src residue to that of chicken, subtract three.
Myristoylation facilitates the attachment of Src to membranes, and myristoylation is required for Src operation in cells [3]. The seven N-terminal amino acids beginning with glycine are required for the myristoylation of Src and v-Src [9,10]. Mutational studies show that a correlation exists between N-myristoylation, subse- quent membrane association, and the ability of v-Src protein kinase to transform cells into a neoplastic state. The catalytic subunit of the serine/threonine protein kinase A (PKA) and the Abl non- receptor protein-tyrosine kinase are myristoylated, but they are largely cytosolic [11,12]. Myristoylation is thereby not sufﬁcient to ensure protein kinase membrane localization.
SH3 domains ( 60 amino acid residues) bind to sequences that can adopt a left-handed helical conformation [13]. The SH3 domain is a þ-barrel consisting of ﬁve antiparallel þ-strands and two promi- nent loops called the RT and n-Src loops (Fig. 2). These loops lie at either end of a surface composed of aromatic and hydrophobic residues that make up the recognition site for protein sequences bearing a PxxP motif. These sequences adopt a polyproline type II helical conformation that complexes with the SH3 domain. The pro- lines interact with aromatic side chains on the SH3 surface. Not all type II left-handed helices contain multiple prolines [13]. For exam- ple, the linker between the Src SH2 domain and kinase domain that interacts with the SH3 domain contains Pro249 in a type II helix. This residue interacts with N138 and Y139 of the SH3 domain of human Src.
SH2 domains ( 100 amino acid residues) bind to distinct amino acid sequences C-terminal to phosphotyrosine [14]. Songyang and Cantley analyzed the binding of a library of phosphopeptides to SH2 domains to deﬁne preferred docking sequences [15]. The SH2 domains of Fgr, Fyn, Lck, and Src select pYEEI in preference to other sequences. X-ray crystallographic studies of the Src SH2 domain indicate that (i) the phosphotyrosine ligand binds to an invariant arginine and (ii) the isoleucine at the P + 3 position binds within a hydrophobic pocket [16]. The acidic residues at the pY + 1 and pY + 2

Fig. 2. Secondary structures of (A) inactive and (B) active Src. The SH3 domain is cyan, and the SH2 domain is magenta. C-t, C-terminus; N-t, N-terminus. The ﬁgures of inactive human Src (A) and active chicken Src (B) were prepared from PDB ID: 2SRC and 3DWQ, respectively.
This ﬁgure and Figs. 4, 5 and 9 were prepared using the PyMOL Molecular Graphics System Version 1.5.0.4 Schrödinger, LLC.

positions of the SH2 binding partner interact with basic residues on the surface of the SH2 domain.
The Src SH2 domain (Fig. 2) consists of a central three-stranded þ-sheet with a single helix packed against each side (a1 and a2). The SH2 domain forms two recognition pockets: one co-ordinates phosphotyrosine and the other binds one or more hydrophobic residues C-terminal to the phosphotyrosine. The phosphotyrosine pocket contains a conserved arginine residue (Arg178 in human Src). The Src SH2 domain, however, can bind to a variety of sequences that do not conform to this optimal pYEEI sequence, and other parts of proteins beyond the vicinity of the phosphotyrosine contribute to the formation of the binding interface. The human Src SH2 domain binds intramolecularly to C-terminal pTyr530 that results in inhibition of protein kinase activity. The sequence of this intramolecular site is pYQPG, which is a nonoptimal Src SH2- binding sequence. As a result, this binding can be readily displaced by more optimal phospholigands that can lead to enzyme activa- tion.
One of the two most important regulatory phosphorylation sites in Src is Tyr530, six residues from the C-terminus. Under basal con- ditions in vivo, 90–95% of Src is phosphorylated at Tyr530 [17], which binds intramolecularly with the Src SH2 domain. SH2 and SH3 binding partners are able to displace the intramolecular asso- ciation that stabilizes the dormant form of the enzyme [3]. The Tyr530Phe mutant is more active than the wild type enzyme and

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4 R. Roskoski Jr. / Pharmacological Research xxx (2015) xxx–xxx

196 can induce anchorage-independent growth in vitro and tumors this is accompanied by the conversion to the closed form as catalysis 260
197 in vivo [18,19]. Tyr530 phosphorylation results from the action occurs. After catalysis, phosphorylated protein and then MgADP are 261
198 of other protein-tyrosine kinases including Csk and Chk [20–22]. released as the enzyme is reconverted to the open form prior to the 262
199 Src undergoes an intermolecular autophosphorylation catalyzed by next catalytic cycle. 263
200 another Src molecule at activation loop Tyr419, which promotes
201 kinase activity [3]. Interconversion of the autoinhibited and active 264
202 The SH2 and SH3 domains have four important functions [3]. conformations of Src 265
203 First, they constrain the activity of the enzyme via intramolecular
204 contacts. Second, proteins that contain SH2 or SH3 binding part- Src regulation by the latch, clamp, and switch 266
205 ners can interact with the SH2 or SH3 domains of Src and attract
206 them to speciﬁc cellular locations. Third, as a result of displacing Early biochemical studies led to the suggestion that the SH2 267
207 the intramolecular SH2 or SH3 domains, proteins lead to the acti- and SH3 domains inhibit Src protein kinase activity by directly 268
208 vation of Src kinase activity. And fourth, proteins containing SH2 or blocking the active site. However, its three-dimensional structure 269
209 SH3 binding partners may preferentially serve as substrates for Src showed that the SH2 and SH3 domains occur in the rear of the 270
210 protein-tyrosine kinase. The group I and II Src-family kinase mem- kinase domain (Fig. 2A). Moreover, the SH2 domain, which binds 271
211 bers contain an N-terminal myristoyl group and the seven-residue to the inhibitory pTyr530, is 40 A˚ from the active site. These struc- 272
212 C-terminal regulatory tail [4]. The group III Src family kinases (Frk, tural studies indicated that the inhibitory effects of SH2 and SH3 273
213 Srm, and Brk) lack the myristoyl group and the seven-residue C- are indirect. 274
214 terminal regulatory tail, but they possess the SH1, SH2, and SH3 The apparatus controlling Src activity has three components that 275
215 domains. Harrison calls the latch, the clamp, and the switch (Fig. 3) [25]. The 276
SH2 domain binds to pTyr530 in the C-terminal tail to form the 277
216 Secondary structure of the Src protein kinase domain: the protein latch, which stabilizes the attachment of the SH2 domain to the 278
217 kinase fold large lobe. The avian oncogenic form of Src, which lacks a tyrosine 279
in its C-terminal tail, and the Tyr530Phe mutant of Src are constitu- 280
218 The small lobe of all protein kinases is dominated by a ﬁve- tively activated [3,4,18,19]. The SH3 domain contacts the small lobe. 281
219 stranded antiparallel þ-sheet (þ1–þ5) and an important regulatory The linker between the SH2 and kinase domains contains proline 282
220 aC-helix (Fig. 2A) [23]. The ﬁrst X-ray structure of a protein kinase at position 249 that is part of a motif that binds to the SH3 domain 283
221 (PKA) [24] contained an aA and an aB-helix proximal to aC (PDB ID: and attaches the SH3 domain to the small kinase lobe. The linker 284
222 1CPK), but these ﬁrst two helices are not conserved in the protein does not possess the classical PxxP signature [13], but this stretch 285
223 kinase family. The active site of the kinase domain occurs within a of residues readily forms a left-handed (polyproline type II) helix. 286
224 cleft that is between the small N-lobe and the large C-lobe. Prior to the determination of the three-dimensional structure of 287
225 The large lobe of the Src protein kinase domain is mainly a- Src, investigators used various algorithms in attempts to identify 288
226 helical with six conserved segments (aD–aI) that occur in all a Src sequence that could bind to an SH3 domain, but these were 289
227 protein kinases (Fig. 2A) [23]. The ﬁrst X-ray structure of a pro- unsuccessful. 290
228 tein kinase (PKA) possessed a short helix between the activation The clamp is an assembly of the SH2 and SH3 domains behind 291
229 segment and the aF-helix, which was not named [24]. However, the kinase domain that functions in concert. As a result of clam- 292
230 this aEF-helix is conserved in all protein kinase structures and rep- ping the SH2 and SH3 domains to the kinase domain, helix aC and 293
231 resents a seventh-conserved segment in the C-lobe. The aF-helix its critical Glu313 are displaced that results in an autoinhibited 294
232 forms an important hydrophobic core. The large lobe of active Src enzyme. A hydrophobic interaction between Trp263 of the SH3 295
233 contains a helix between the aH and aI segment (aHI) (Fig. 2A). The kinase linker and Gln315 of the aC-helix participates in its dis- 296
234 activation segment of inactive Src, which contains the aAL1, aAL2, placement producing an autoinhibited enzyme (not shown). The 297
235 and aEF-helices, is compact while that of active Src is an extended switch refers to the kinase-domain activation loop; the activation 298
236 open loop lacking aAL1 and aAL2, but still containing the aEF-helix. loop can switch from an inactive to active conformation follow- 299
237 The large lobe of active Src kinase contains seven short þ-strands ing its autophosphorylation at Tyr419 as catalyzed by a partner Src 300
238 (þ6–þ12) (Fig. 2B). The þ6-strand, the primary sequence of which molecule. 301
239 occurs before the catalytic loop, interacts with the activation seg-
240 ment þ9-strand. The þ7-strand interacts with the þ8-strand, the Unlatching by phosphatases 302
241 primary structures of which occur between the catalytic loop and
242 the activation segment. The kinase domain of PKA and most active Dormant Src exists in equilibrium with pTyr530 bound to or free 303
243 protein kinases contain these nine þ-strands. However, the active from the SH2 domain with the bound state greatly favored. When 304
244 Src kinase domain contains three additional strands (þ10–12). The pTyr530 is displaced from the SH2-binding pocket, the protein can 305
245 þ10-strand from the activation segment interacts with the þ11- be unlatched with the clamp no longer locking the catalytic domain 306
246 strand that occurs proximal to the aF-helix. The þ12-strand occurs in an inactive conformation [25,26]. Furthermore, dissociation of 307
247 in the initial part of the C-lobe immediately after the hinge residues pTyr530 allows dephosphorylation by various protein-tyrosine 308
248 and it interacts with the þ7-strand. Inactive Src contains the þ7 and phosphatases that lead to the unlatched and active enzyme (Fig. 3). 309
249 þ8-strands (not shown), but it lacks the þ6 and þ9–12 strands. Candidate pTyr530 phosphatases include cytoplasmic PTP1B, Shp1 310
250 Note that the þ-strand nomenclature follows that of PKA while (Src homology 2 domain-containing tyrosine phosphatase-1) and 311
251 additional strands in protein kinases are assigned arbitrarily. Shp2 and transmembrane enzymes including CD45, PTPa, PTPs, 312
252 There are two general kinds of conformational motions associ- and PTPh [27]. 313
253 ated with all protein kinases including those of the Src family; one
254 involves conversion of an inactive conformation into a catalytically Switching and unclamping 314
255 competent form. Activation typically involves changes in the orien-
256 tation of the aC-helix in the small lobe and the activation segment Following the unlatching of Src as catalyzed by various protein- 315
257 in the large lobe. The active kinase then toggles between open and tyrosine phosphatases, Tyr419 can then undergo autophosphory- 316
258 closed conformations as it goes through its catalytic cycle. The open lation by another Src kinase molecule in a process called switching 317
259 form of the active enzyme binds MgATP and the protein substrate; (Fig. 3). Following autophosphorylation, the enzyme is stabilized 318

R. Roskoski Jr. / Pharmacological Research xxx (2015) xxx–xxx 5

Fig. 3. The Src latch, clamp, and switch. Unlatching, unclamping, and switching lead to the formation of active Src.

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in its active state. Exogenous substrates decrease autophospho- rylation in vitro during activity measurements [28]. This ﬁnding is consistent with the notion that activation loop phosphoryla- tion occurs in trans and involves two kinase molecules in an intermolecular reaction and not a cis intramolecular reaction, which is less likely to be inhibited by competition with exogenous substrates.
The structural design of Src allows for its regulation at multiple levels including competition between intramolecular and external binding partners [25]. The intramolecular interactions maintain an inactive state and external interactions promote an active state. Proteins that bind to the Src SH2 domain, the SH3 domain, or both disrupt the clamp, activating the kinase (Fig. 3).

Conversion of active to inactive Src

To return to the immobilized inactive state, the activation seg- ment phosphate (pY419) is liberated by PTB-BAS [27] and the intramolecular SH2/SH3 binding partners replace the intermolec- ular binding partners. Human PTP-BAS is a ubiquitously expressed cytosolic phosphatase that contains a FERM domain, ﬁve PDZ domains, and a PTP domain. FERM is the acronym of four point one/ezrin/radixin/moesin. FERM proteins associate with F-actin and with the plasma membrane. PDZ domains are modular pro- tein interaction domains of 80–90 residues in signaling proteins that bind to the C-terminus of other speciﬁc proteins. PTP-BAS was derived initially from human white blood cell basophils (BAS refers to basophil) and BL (mouse) refers to basophil like. PTP-BAS co-localizes in membrane fractions with Src family kinases.
PTP-BL is the mouse homologue of human PTP-BAS. Palmer
et al. found that the bacterially expressed mouse PTP-BL phos- phatase domain, but not a catalytically inactive mutant, catalyzes the dephosphorylation of mouse Src speciﬁcally at pTyr419 [29]. In contrast, this enzyme does not act upon pTyr530. These investiga- tors found that ephrinB2, an important regulator of morphogenesis, leads to the activation of Src kinase in mouse NIH3T3 cells. Activa- tion is apparent at 10 min and is absent at 30 min. The deactivation is associated with the recruitment of PTP-BL and dephosphory- lation of Src pTyr419. As noted above, pTyr419 occurs in the activation segment and is associated with increased Src activity. It is possible that other phosphatases are involved in regulatory pTyr419 dephosphorylation.

Csk or Chk catalyze the phosphorylation of Tyr530 so that the latch can reform. Csk, a cytoplasmic protein-tyrosine kinase, was the ﬁrst enzyme discovered that catalyzes the phosphorylation of the regulatory C-terminal tail tyrosine of Src (Fig. 3). Okada and Nakagawa isolated this enzyme from neonatal rat brain and demonstrated that it catalyzes the phosphorylation of Src at Tyr530 [20]. Following phosphorylation, the Km of Src for ATP and for acid-denatured enolase is unchanged, but the kcat is decreased 50%. Using puriﬁed Src, the activity of the Tyr530 phosphorylated enzyme in vitro ranges from 0.2–20% that of the unphosphorylated enzyme depending upon the experimental conditions.
Chk is a second enzyme that catalyzes the phosphoryla- tion of the inhibitory tyrosine of Src-family kinases [22]. Csk is expressed in all mammalian cells, whereas Chk is limited to breast, hematopoietic cells, neurons, and testes [3]. Csk and Chk consist of an SH3, SH2, and kinase domain; these enzymes lack the N-terminal myristoyl group and the C-terminal regulatory tail phosphoryla- tion site found in Src [30]. Besides inactivating Src by catalytic phosphorylation, Chk forms a noncovalent inhibitory complex with Src. The association of Chk with the activated and autophosphory- lated form of Src inhibits Src kinase activity [31]. The action of Chk thereby overrides that of Src. Chk can also bind to unphosphory- lated Src and prevent its activation segment phosphorylation.
There are four possible Src enzyme forms: (i) nonphospho-
rylated, (ii) Tyr530 phosphorylated, (iii) Tyr419 phosphorylated, and (iv) both Tyr530 and 419 phosphorylated enzymes. Src with phosphorylated Tyr530 cannot undergo autophosphorylation; the residue must ﬁrst be dephosphorylated. However, Src with Tyr419 autophosphorylation is a substrate for C-terminal Src kinase, but the doubly phosphorylated enzyme is active, so that Tyr419 phosphorylation overrides inhibition produced by Tyr530 phos- phorylation [32].

Structure of the Src kinase domain skeleton

The regulatory spine

Taylor and Kornev [33] and Kornev et al. [34] analyzed the struc- tures of active and inactive conformations of about two dozen protein kinases and determined functionally important residues by a local spatial pattern (LSP) alignment algorithm. In contrast to the protein kinase amino acid signatures noted later such as DFG

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6 R. Roskoski Jr. / Pharmacological Research xxx (2015) xxx–xxx

398 or HRD, the residues that constitute the spines were not identiﬁed including the Src family, form hydrophobic contacts with the aF- 428
399 by sequence analyses per se. Rather, they were identiﬁed by their helix [34]. 429
400 three-dimensional location based upon a comparison of the X-ray Src has been observed in both inactive (PDB ID: 2SRC) and 430
401 crystallographic structures [33,34]. active (PDB ID: 3DQW) conformations by X-ray crystallography. 431
402 The local spatial pattern alignment analysis revealed a skele- The active form of the chicken enzyme, which contains a Thr to 432
403 ton of four nonconsecutive hydrophobic residues that constitute Ile gatekeeper mutation, was chosen instead of an active wild type 433
404 a regulatory or R-spine and eight hydrophobic residues that con- human enzyme (e.g., 1Y57) because the chicken enzyme contains 434
405 stitute a catalytic or C-spine (Fig. 4C and D). The R-spine interacts (i) an activation segment phosphotyrosine and (ii) a bound ATP-μ- 435
406 with a conserved aspartate (D447) in the aF-helix. As noted later in S that were needed for analyses as described later. Although the 436
407 this section, there are three conserved “shell” residues that interact kinase domains of the active and autoinhibited enzyme are nearly 437
408 with the R-spine. Altogether each protein kinase contains 16 amino superimposable, the aC-helices and the activation segments of the 438
409 acids that make up this protein kinase skeletal assembly. Each spine active and inactive enzyme forms differ from one another (root 439
410 consists of residues derived from both the small and large lobes. mean square deviation > 6 A˚ ). Note the subluxation, or kink, at the 440
411 The regulatory spine contains residues from the activation segment RS3 residue of the Src regulatory spine in inactive Src (Fig. 4D and 441
412 and the aC-helix, whose conformations are important in deﬁning F). This abnormality is associated with the aC out and catalytically 442
413 active and inactive states. The catalytic spine governs catalysis by inactive structure of the Src kinase domain. 443
414 facilitating ATP binding. The two spines dictate the positioning of
415 the protein substrate (R-spine) and ATP (C-spine) so that cataly-
416 sis results. The proper alignment of the spines is necessary for the The catalytic spine 444
417 assembly of an active kinase. The catalytic spine of protein kinases consists of residues from 445
418 The Src regulatory spine consists of a residue from the begin- the small and large lobes and is completed by the adenine base 446
419 ning of the þ4-strand (Leu328, human Src residue number), from of ATP [33,34]. The two residues of the small lobe of the Src pro- 447
420 the C-terminal end of the aC-helix (Met317), the phenylalanine tein kinase domain that bind to the adenine group of ATP include 448
421 of the activation segment DFG (Phe408), along with the HRD- Val284 from the beginning of the þ2-strand and Ala296 from the 449
422 histidine (His387) of the catalytic loop. Met317 and comparable conserved Ala-Xxx-Lys of the þ3-strand. Furthermore, Leu396 from 450
423 residues from other protein kinases are four residues C-terminal to the middle of the large lobe þ7-strand binds to the adenine base in 451
424 the conserved aC-glutamate. The backbone of His387 is anchored the active enzyme. Val284, Ala296, and Leu396 characteristically 452
425 to the aF-helix by a hydrogen bond to a conserved aspartate make hydrophobic contacts with the scaffolds of ATP-competitive 453
426 residue (Asp447). The protein-substrate positioning segment, the small molecule inhibitors. Ile395 and Val397, hydrophobic residues 454
427 activation segment, and the aHI-loop of protein kinase domains, that ﬂank Leu396, bind to Leu349 at the beginning of the aD-helix. 455

Fig. 4. Overview of the structure of the (A) active and (B) inactive Src kinase domain. Location of the C- and R-spines of (C) active and (D) inactive Src. (E) Superposition of active and inactive Src and (F) their C- and R-spines. Important salt bridges (SB) and hydrogen bonds (HB) in (F) active and (G) inactive Src. AL, activation loop; AS, activation segment; H8I, hydrophobic interaction.
The ﬁgures of active chicken Src were prepared from PDB ID: 3DQW and inactive human Src from PDB ID: 2SRC.

R. Roskoski Jr. / Pharmacological Research xxx (2015) xxx–xxx 7

Table 1
Src and PKA R-spine (RS), R-shell (Sh), and C-spine residues.
Symbol Chicken Src Human Src Murine PKAa
Regulatory spine
þ4-strand (N-lobe) RS4 Leu325 Leu328 Leu106
C-helix (N-lobe) RS3 Met314 Met317 Leu95
Activation loop F of DFG (C-lobe) RS2 Phe405 Phe408 Phe185
Catalytic loop His or Tyr (C-lobe)b RS1 His384 His387 Tyr164
F-helix (C-lobe) RS0 Asp444 Asp447 Asp220
R-shell
Two residues upstream from the gatekeeper Sh3 Ile336 Ile339 Met118
Gatekeeper, end of þ4-strand Sh2 Thr338 Thr341 Met120
aC-þ4 loop Sh1 Val323 Val326 Val104
Catalytic spine
þ2-strand (N-lobe) Val281 Val284 Val57
þ3-AXK motif (N-lobe) Ala293 Ala296 Ala70
þ7-strand (C-lobe) Leu393 Leu396 Leu173
þ7-strand (C-lobe) Ile392 Ile395 Leu172
þ7-strand (C-lobe) Val394 Val397 Ile174
D-helix (C-lobe) Leu346 Leu349 Met128
F-helix (C-lobe) Leu451 Leu454 Leu227
F-helix (C-lobe) Leu455 Leu458 Met231
a From Ref. [34,35].
b Part of the HRD (His-Arg-Asp) or YRD (Tyr-Arg-Asp) sequence.

456 The aD-helix Leu349 binds to Leu454 and Leu458 in the aF-helix. access to a hydrophobic pocket adjacent to the adenine binding 476
457 Note that both the R-spine and C-spine are anchored to the aF- site [36,37] that is occupied by portions of many small molecule 477
458 helix, which is a very hydrophobic component of the enzyme that inhibitors as described later. Using the previous local spatial pat- 478
459 is entirely within the protein and not exposed to the solvent. The tern alignment data [34], only three of 14 amino acid residues in 479
460 aF-helix serves as a sacrum that supports the spines, which in turn PKA surrounding RS3 and RS4 are conserved, and these are the 480
461 anchor the protein kinase catalytic muscle. Table 1 lists the residues shell residues that serve as collateral spinal ligaments that stabi- 481
462 of the spines of human and chicken Src and the catalytic subunit of lize the protein kinase vertebral column or spine [35]. The V104G 482
463 murine PKA. mutation (Sh1) decreased the catalytic activity of PKA by 95%. The 483
M120G (Sh2) and M118G (Sh3) double mutant was devoid of cat- 484
464 Spinal collateral ligaments or shell residues alytic activity. These results provide evidence for the importance of 485
the shell residues in stabilizing the spine and maintaining protein 486
465 Using site-directed mutagenesis and sensitive radioisotopic kinase activity. 487
466 enzyme assays, Meharena et al. identiﬁed three residues in murine A comparison of the active and inactive Src R-spines shows 488
467 PKA that stabilize the R-spine, and they referred to them as shell that RS3 of the dormant enzyme is displaced when compared with 489
468 residues [35]. Going from the connecting aspartate at the bottom active Src, a result that is consistent with the displaced aC-helix of 490
469 in the aF-helix up to the spine residue in the þ4-strand at the top, the inactive enzyme (Fig. 5). The RS3 and RS4 a-carbon atoms of 491
470 these investigators labeled the regulatory spine residues RS0, RS1, the active and inactive kinase domains differ in location by 2.6 A˚ 492
471 RS2, RS3, and RS4 (Fig. 5 and Table 1). The three shell residues are and 1.2 A˚ , respectively, and the terminal methyl carbon atoms of 493
472 labeled Sh1, Sh2, and Sh3. Sh3 interacts with RS4, and Sh1 interacts Met317 (RS3) differ in location by 6.2 A˚ . The Sh1 and Sh2 residues of 494
473 with RS3 and Sh2. Sh2, which is the classical gatekeeper residue, active and autoinhibited Src are nearly superimposable while the 495
474 interacts with Sh1 below it and with RS4 next to it. The term gate- a-carbon atoms of Sh3 are modestly displaced (1.9 A˚ ). Moreover, 496
475 keeper refers to the role of such residues in allowing or disallowing the C-spines of active and inactive Src are nearly superimposable. 497

Fig. 5. The Src regulatory and catalytic spines and shell residues. (A) Interaction of the shell (Sh) residues with those of the regulatory spine (RS). The R-spine is depicted as spherical CPK residues and the shell residues are shown as sticks in (B) active and (C) inactive Src. (D) Superposition of the (i) spine residues and (ii) shell residues from active (PDB ID: 3DQW in blue) and inactive (PDB ID: 2SRC in red) Src.

8 R. Roskoski Jr. / Pharmacological Research xxx (2015) xxx–xxx

498

Since the RS3 residues of the R-spine of active and inactive enzyme

Table 2
Important structural and functional residues in Src.

499 forms differ in location, it is natural to expect that the surrounding

500

501

502

503

504

505

506

507

508

509

shell residues will also vary in their three-dimensional location. The 18 residues of the Src family skeletal assembly are con-
served. Of the group I (Src, Yes, Fyn, Fgr) and group II (Blk, Lck, Lyn, Hck) members of the Src family, nearly 97% of the skeletal assembly residues are identical. This compares with only 70% identity of the kinase domain residues (Src 270–523) in the eight Src kinase fam- ily members. The four nonidentical C-spine residues correspond to Src Leu458 within the hydrophobic aF-helix. These residues are all leucine in the group I members of the Src family, but consist of valine (Blk) or isoleucine (Lck, Lyn, Hck) in the group II Src fam-

Chicken Src Human Src
SH3 domain 81–142 84–145
SH2 domain 148–245 151–248
SH1 catalytic domain 267–520 270–523
N-lobe
Glycine-rich loop: GQGCFG 274–279 277–282
þ-3 lysine (K of K/E/D/D) K295 K298
aC-Glu (E of K/E/D/D) E310 E313

aC-helix þ5-strand H8 interaction L308–I336 L311–I339
aC-þ4 loop and aE helix H8 interaction L322–Y376 L325–Y379 Hinge residues: EYMSKG 339–344 342–347
C-lobe

510

511

ily. The evolutionary conservation of the Src family kinase domain skeletal assembly underscores its importance.

aE-activation segment loop and activation segment H8 interaction

Y382–L410 Y385–L413

Catalytic loop HRD (ﬁrst D of K/E/D/D) 386 389
Intracatalytic loop hydrogen bond R388–N391 R391–N394

512

Src catalytic residues

Catalytic loop–activation segment hydrogen bonds

R385–pY416; N391–D404

R388–pY419; N394–D407

Catalytic loop N 391 394

513

Properties of the small and large lobes

Activation segment DFG (second D of K/E/D/D)

404 407

514

Like all protein kinases, the Src protein kinase domain has a

Activation segment 404–432 407–435
Mg2+-positioning loop:DFGLAR 404–409 407–412

515

516

517

518

519

small amino-terminal lobe and large carboxyterminal lobe (Fig. 4A and B) ﬁrst described by Knighton et al. for PKA [24]. The two lobes form a cleft that serves as a docking site for ATP. The small lobe of protein kinases contains a conserved glycine-rich (GxGx8G) ATP- phosphate–binding loop, which is the most ﬂexible part of the lobe.

Activation segment tyrosine phosphorylation site

416 419

520 The glycine-rich loop is near the phosphates of the ATP substrate
521 as described later. The þ1 and þ2-strands of the N-lobe harbor the

522

523

524

525

526

527

528

529

530

531

532

533

534

535

536

537

538

539

540

541

542

543

544

545

546

547

548

549

550

551

552

553

554

555

556

557

558

559

adenine component of ATP and they interact with ATP-competitive small molecule inhibitors. The þ3-strand typically contains an Ala- Xxx-Lys sequence, the lysine of which in Src (K298) forms a salt bridge with a conserved glutamate near the center of the aC-helix (E313) of protein kinases. The presence of a salt bridge between the þ3-lysine and the aC-glutamate is a prerequisite for the formation of the activate state and corresponds to the “aC-in” conformation (Fig. 4G). By contrast, Lys298 and Glu313 of the dormant form of Src fail to make contact and this structure corresponds to the dis- placed “aC-out” conformation (Fig. 4H). The aC-in conformation is necessary, but not sufﬁcient, for the expression of full kinase activity.
The large lobe contains a mobile activation segment with an extended conformation in active enzymes and closed conformation in dormant enzymes. The ﬁrst residues of the activation segment of protein kinases consist of DFG (Asp-Phe-Gly). The DFG exists in two different conformations in the protein kinase family. In the dormant activation segment conformation of many protein kinases, the aspartate side chain of the conserved DFG sequence faces away from the active site. This is called the “DFG-Asp out” conforma-
tion. In the active state, the aspartate side chain faces into the ATP-binding pocket and coordinates Mg2+. This is called the “DFG- Asp in” conformation. This terminology is better than “DFG-in” and “DFG-out” because, in the inactive state, the DFG-phenylalanine may move into the active site (while the DFG-aspartate moves out) [38]; it is the ability of aspartate to bind (Asp-in) or not bind (Asp- out) to Mg2+ in the active site that is crucial. However, the inactive conformation of the Src kinase activation segment exists in a closed conformation but with the DGF-Asp directed inward. The distinc- tive aC out and DGF-Asp in combination is labeled as the Src family
kinase-like inactive conformation. The Mg2+-positioning segment
(Fig. 4C) of Src consists of the ﬁrst ﬁve residues of the activation segment (DFGLA).
The activation segments of protein kinases including Src typ- ically ends with APE (Ala-Pro-Glu). The last eight residues of the activation segment of Src are PIKWTAPE, which make up the protein-substrate positioning segment (Fig. 4A and B). The R-group of the ﬁrst proline in this sequence serves as a platform that

interacts with the tyrosyl residue of the peptide/protein substrate that is phosphorylated [39]. In protein-serine/threonine kinases, the serine or threonine interacts with backbone residues near the end of the activation segment and not with an R-group. The acti- vation segment of Src contains a phosphorylatable tyrosine and its phosphorylation, like that of most other protein-tyrosine kinases [34], is required for enzyme activation [27]. As noted previously, Harrison referred to this phosphorylation as switching [25].
Two conserved hydrophobic interactions in Src and other pro- tein kinases contribute to kinase domain stability. A hydrophobic contact between Leu311, which is two residues N-terminal to the Glu313 in the aC-helix, with Ile339 near the N-terminus of the þ4- strand helps to stabilize the N-terminal lobe. Moreover, another hydrophobic contact from Leu325 in the aC-þ4 loop of the small lobe and Tyr379 near the carboxyterminal end of the aE-helix in the large lobe (eight residues upstream from HRD of the catalytic loop) further stabilizes the interaction between the two lobes (Fig. 4A).

The K/E/D/D protein kinase signature

The Src kinase domain consists of the characteristic bilobed pro- tein kinase architecture [26,40–42]. Residues 270–341 make up the small amino-terminal lobe of the kinase; residues 348–523 make up the large carboxyterminal lobe (Fig. 4A). As described for PKA [33], ATP binds in the cleft between the small and large lobes of Src and the protein substrate binds to the larger carboxyterminal lobe. Furthermore, active site residues are derived from both the small and large lobes of the kinase and changes in the orientation of the two lobes can promote or restrain activity.
Hanks et al. identiﬁed 11 subdomains with conserved amino acid residue signatures that constitute the catalytic core of protein kinases [43]. Of these, the four following residues, which constitute a K/E/D/D motif, illustrate the inferred catalytic properties of Src. Lys298 (the K of K/E/D/D) represents an invariant residue of protein kinases that forms ion pairs with the þ- and μ-phosphates of ATP and with Glu313 in the aC-helix (the E of K/E/D/D) (Table 2). Asp389 orients the tyrosyl group of the substrate protein in a catalytically

560

561

562

563

564

565

566

567

568

569

570

571

572

573

574

575

576

577

578

579

580

581

582

583

584

585

586

587

588

589

590

591

592

593

594

R. Roskoski Jr. / Pharmacological Research xxx (2015) xxx–xxx 9

595 competent state. Asp389 functions as a base that abstracts a proton His87 of the aC-helix, (ii) Arg165 of the catalytic loop H/YRD, (iii) 659
596 from tyrosine thereby facilitating its nucleophilic attack of the μ- Tyr215 in the aEF/aF-loop, and (iv) Lys189 and Thr195 within the 660
597 phosphorus atom of MgATP; Asp389 (the ﬁrst D of K/E/D/D) is called activation segment [33]. The glutamate at the end of the activation 661
598 the catalytic base. This base occurs within the catalytic loop (Fig. 4A segment forms a conserved salt bridge with Arg280 in the aHI-loop. 662
599 and B) that generally has the sequence HRDLRAAN in non-receptor Within the R-spine, Phe185 of DFG within the PKA activation seg- 663
600 protein-tyrosine kinases including Src. Asp407 is the ﬁrst residue ment makes hydrophobic contacts with Leu95 of the aC-helix and 664
601 of the activation segment found in the large lobe (the second D of with Tyr164 of the catalytic loop (equivalent to the histidine HRD 665
602 K/E/D/D). Asp407 binds Mg2+, which in turn coordinates the þ- and of most protein kinases). 666
603 μ-phosphate groups of ATP. The activation segment Tyr419-phosphate of Src differs from 667
604 The small and large lobes can adopt a range of relative orienta- the Thr197-phosphate of PKA in its interactions within the kinase 668
605 tions, opening or closing the active site cleft [26,44]. Within each domain. The Src Tyr419-phosphate does not interact with any 669
606 lobe is a polypeptide segment that has an active and an inactive residues within the aC-helix, but it does form a salt bridge with 670
607 conformation. In the small lobe, this segment is the aC-helix [44]. Arg388 of the catalytic group HRD (Fig. 4 G). The Src Tyr419- 671
608 The aC-helix in some kinases rotates and translates with respect phosphate forms a salt bridge with Arg412 within the activation 672
609 to the rest of the lobe, making or breaking part of the active cat- segment, but it fails to make contact with residues within the 673
610 alytic site. In the large lobe, the activation segment adjusts to make aEF/aF-loop. Like PKA, Src Glu435 at the end of its activation seg- 674
611 or break part of the catalytic site. In most protein kinases, phos- ment forms a salt bridge with Arg509 that lies within its aHI-loop. 675
612 phorylation of a residue within the activation segment stabilizes The DFG-Phe407 makes a hydrophobic contact with Met317 of the 676
613 the active conformation; in human Src, this residue corresponds to N-lobe aC-helix and the HRD-His387 of the C-lobe catalytic loop as 677
614 Tyr419. part of the R-spine. Also, like PKA, the activation segment þ9-strand 678
615 The structures of active and dormant Src kinases including the interacts with the þ6-strand near the catalytic loop. The activation 679
616 SH3, SH2, and SH1 (kinase) domains have been solved by X-ray segment þ10-strand interacts with the þ11-strand just proximal to 680
617 crystallography [26,40–42]. The conformation of the activation the aF-helix; however, this interaction is lacking in PKA. Thus, the 681
618 loop differs between active and inactive kinases [44]. In protein stabilization of the Src activation segment differs in detail from that 682
619 kinases that are inactive, the activation loop has various com- observed in PKA. Moreover, the þ6 and þ9-strand and the þ10 and 683
620 pact conformations. In structures of enzymes that are in an active þ11-strand interactions are lacking in the inactive conformations 684
621 state, the activation loop is in an extended conformation. There are of Src. 685
622 two crucial aspects to this active conformation. First, the aspar- Hydrophobic interactions occur within the Src activation seg- 686
623 tate residue (Asp407 in Src) within the conserved DFG motif at ment involving (i) Phe407 (the Phe of DFG) and Leu410 and (ii) 687
624 the amino-terminal base of the activation segment binds to the Phe427, Ala425, and Phe442. However, these hydrophobic inter- 688
625 magnesium ion as noted above. Second, the rest of the loop is pos- actions occur in both (i) active (PDB ID: 3DQW) and (ii) inactive 689
626 itioned away from the catalytic center in an extended conformation Src (PDB ID: 2SRC); accordingly, they fail to explain any additional 690
627 so that the C-terminal portion of the activation segment provides stabilization of the activation segment in its active conformation. 691
628 a platform for protein substrate binding. Using molecular dynamics simulations, Meng and Roux reported 692
629 In dormant Src kinase, residues 410–413 and 417–421 of the that phosphorylation of the activation loop tyrosine of Src helps to 693
630 activation segment form short a-helices (aAL1 and aAL2) (Fig. 4B). stabilize the R-spine and the HRD motif [46]. They conclude that 694
631 As a result, aAL1 displaces the aC-helix into its inactive out confor- this phosphorylation helps to lock the enzyme into its catalytically 695
632 mation so that Glu313 in the helix cannot form a critical salt bridge active conformation. 696
633 with Lys298. The aAL2-helix helps to bury the side chain of Tyr419 Knowledge of the active and inactive conformations of protein 697
634 (the site of activating phosphorylation). The aAL1 and aAL2-helices kinases can serve as an aid in drug discovery [38]. Although the 698
635 are thus important autoinhibitory components. They (i) preclude tertiary structure of catalytically active protein kinase domains is 699
636 protein/peptide substrate recognition, (ii) sequester Tyr419, and strikingly similar, Huse and Kuriyan reported that the crystal struc- 700
637 (iii) stabilize the inactive conformation of the kinase domain [26]. tures of inactive enzymes reveal a multitude of distinct protein 701
638 The interconversion of the inactive and active forms of Src kinase conformations [44]. The practical consequence of this is that 702
639 kinase also involves an intricate electrostatic switch. In the dormant drugs targeting speciﬁc inactive conformations may be more selec- 703
640 enzyme, the þ4-lysine (K298) forms a salt bridge with the DFG-Asp tive than those targeting the active conformation [47]. Huse and 704
641 (D407) residue, and the aC-Glu313 forms a salt bridge with Arg412 Kuriyan noted that protein kinases usually assume their less active 705
642 (the sixth residue of the activation segment). Moreover, the–NH of conformation in the basal or non-stimulated state and the acquisi- 706
643 Asn394 hydrogen bonds with a carboxylate of Asp389 (the D of tion of their activity may involve several layers of regulatory control 707
644 HRD). The conversion to the active enzyme form entails an elec- [44]. 708
645 trostatic switch: the þ4-lysine (K298) now forms a salt bridge with
646 the aC-Glu (E313) with the concomitant formation of the aC-in
647 conformation and the–NH of Asn394 now hydrogen bonds with a Role of magnesium ions in the protein kinase catalytic 709
648 carboxylate of Asp407 (the D of DFG). Following the phosphory- process 710
649 lation of Tyr419 in the activation segment, the phosphate forms
650 salt bridges with Arg388 within the catalytic loop and with Arg412 Participation of two magnesium ions in catalysis 711
651 within the activation segment (Fig. 4G and H).
Nearly all protein kinases require a divalent cation such as Mg2+

712
652 Stabilizing the Src activation segment or Mn2+ for expression of their activity. Because the cellular con- 713
tent of Mg2+ is much greater than that of Mn2+, Mg2+ is considered 714
653 The phosphorylation of one or more residues in the activation to be the physiologically important cation. The magnesium ion 715
654 segment of the majority of protein kinases is required to gener- plays a dual role in protein kinase reactions. First, the physiologi- 716
655 ate their active conformation. In the case of the catalytic subunit cal nucleotide substrate is MgATP. Second, another magnesium ion 717
656 of murine PKA, this corresponds to the phosphorylation of acti- interacts with the enzyme/metal-nucleotide complex to increase 718
657 vation loop Thr197 as catalyzed by PKA [45]. This activation loop the catalytic efﬁciency (kcat/KMgATP), where KMgATP is the Km for 719
658 phosphate interacts with four different sections of PKA including (i) ATP. 720

10 R. Roskoski Jr. / Pharmacological Research xxx (2015) xxx–xxx

721

722

723

724

725

726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

747

748

749

750

751

752

753

754

755

756

757

The kcat/KMgATP is an apparent second-order rate constant (M−1 s−1) that relates the reaction rate to the concentration of free, rather than total, enzyme (the present discussion ignores the pro- tein substrate of protein kinases). At low substrate concentrations most of the enzyme is free and the reaction velocity is given by v = [Enzyme][MgATP] kcat/KMgATP [48]. This constant (kcat/KMgATP) is called the speciﬁcity constant when it is used to compare the effectiveness of multiple substrates for a given enzyme. For enzyme reactions that are limited only by the rates of diffusion of the enzyme and substrate, the upper limit of the value for kinetic efﬁ- ciency is 108 M−1 s−1. In contrast, the kcat/KMgATP for avian Src [49] is 2310 M−1 s−1, that for human Csk [49] is 500 M−1 s−1, and that for the catalytic subunit of bovine PKA [50] is 2.3 105 M−1 s−1 where the citations denote the sources of data upon which the calcula- tions are based. Unlike general metabolic enzymes, protein kinases function as dynamic molecular switches that are turned on or off. Thus, protein kinases are not continuously active as, for example, metabolic enzymes such as hexokinase [23]. Protein kinases are able to perform their physiological functions despite having low catalytic efﬁciencies.
The function of Mg2+ and other divalent cations in protein
kinase-mediated reactions is intricate. The role of Mg2+ has been examined in numerous receptor and non-receptor protein- serine/threonine and protein-tyrosine kinases (Table 3). Variable effects on the kcat and KMgATP have been observed in response to increasing the concentration of magnesium ion. For Src, Csk and leucine-rich repeat kinase-2 (LRRK2), the kcat is increased whereas the KMgATP is unchanged. For CDK5, ERK2, interleukin-1 receptor associated kinase-4 (IRAK-4) and FGFR1, the kcat is increased and the KMgATP is decreased. For the insulin and Fps receptor protein- tyrosine kinases, the kcat is unchanged and the KMgATP is decreased. And for the catalytic subunit of PKA and for CDK2, we have the unusual situation where both the kcat and the KMgATP are decreased. In all of these cases, however, the kinetic efﬁciency (kcat/KMgATP) increases at higher [Mg2+]. Studies with Bruton’s protein-tyrosine kinase, EGFR, ErbB2, ErbB3, Yes, and VEGFR2 also indicate that the kcat/KMgATP is increased, but studies on the kcat and KMgATP as a

or for Mg2+ (both about 1.6 mM) [62]. They observed that increas- ing the Mn2+ concentration ﬁrst increases the reaction rate, but further increases (Mn2+ > 75 µM) lead to a decline. They suggested that the more tightly bound Mn2+ is an essential metal ion activa- tor while the more weakly bound Mn2+ is an inhibitor of catalytic activity.
Our steady-state kinetic analysis of bovine PKA indicated that a high (10 mM) Mg2+ concentration resulted in a kcat that is about one-ﬁfth that at low (0.5 mM) Mg2+ concentration. Even though these studies led to the terminology of an inhibitory Mg2+ site, the catalytic efﬁciency (kcat/KMgATP) at a high Mg2+ concentration increased by 13-fold as a result of the decreased KMgATP. It is impor- tant to note that in the absence of a nucleotide, divalent cation binding afﬁnity to PKA is very weak [62]. This indicates that both metal binding sites are greatly augmented by ADP/ATP. We found that MgATP and peptide substrate bound randomly to PKA, but the release of product was ordered (phosphopeptide before MgADP) [50].
Zheng et al. determined the X-ray crystal structure of the cat- alytic subunit of murine PKA bound to Mg2+, ATP, and a heat-stabile protein kinase inhibitor that mimics a protein substrate [63]. Crys- tals were prepared under conditions of low [Mg2+] and high [Mg2+]. They observed that MgATP is found between the small and large lobes. Under low [Mg2+] conditions, a single Mg2+ is bound to the þ and μ-phosphates and to the aspartate of the DFG sequence; this magnesium ion is labeled 1: Mg2+ (1). Under high [Mg2+] con- ditions, a second Mg2+ is bound to the a and μ-phosphates and to the asparagine amide nitrogen within the catalytic loop down- stream from the Y/HRD conserved sequence of the catalytic loop. This magnesium ion is labeled 2: Mg2+(2).
The role of each Mg2+ has been the subject of numerous stud- ies during the past two decades. Initially, many investigators thought that Mg2+(1) was the key divalent cation required for the protein kinase reaction [64]. More recently, Jacobsen et al. used steady-state kinetics, X-ray crystallography, and molecular dynamics simulations to investigate the role of two cations in the CDK2-mediated reaction [57]. They demonstrated that two Mg2+

765

766

767

768

769

770

771

772

773

774

775

776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

793

794

795

796

797

798

799

800

801

758 function of [Mg2+] were not reported. ions are essential for efﬁcient phosphoryl transfer. Their studies 802
759 The initial studies on the role of divalent cations on the protein showed that ADP phosphate mobility is more restricted when ADP 803
760 kinase reaction were performed with PKA and the results are now is bound to two Mg2+ ions when compared to one. The cost that 804
761 seen to be somewhat atypical. Using nuclear magnetic resonance is paid to accelerate the chemical process is the limitation in the 805
762 and steady-state kinetic studies, Armstrong et al. observedthat the velocity of ADP release, which is the rate-limiting step in the over- 806
763 catalytic subunit of PKA in the presence of a nucleotide such as ADP all process [65,66]. Jacobsen et al. provide evidence that Mg2+(1) is 807
764 contains two binding sites for Mn2+ (Kd = 6–10 µM and 50–60 µM) released prior to ADP-Mg2+(2) [57]. 808

Table 3
Effect of high Mg2+ concentrations on steady-state kinetic parameters of various protein kinases.
Enzymea Class Speciﬁcity Substrateb kcat KMgATP Kcat/KMgATP References

Chicken Src Non-receptor Tyr Poly-E4 Y ↑ No O ↑ [49] Human Csk Non-receptor Tyr Poly-E4 Y ↑ No O ↑ [49] Human LRRK2 Non-receptor Ser/Thr Peptide I ↑ No O ↑ [51] Human CDK5 Non-receptor Ser/Thr Peptide II ↑ ↓ ↑ [52] Rat ERK2 Non-receptor Ser/Thr Ets138 ↑ ↓ ↑ [53] Human IRAK-4 Receptor Ser/Thr Peptide III ↑ ↓ ↑ [54] Xenopus FGFR-1 Receptor Tyr Poly-E4 Y ↑ ↓ ↑ [49] Rat insulin receptor Receptor Tyr Poly-E4 Y No O ↓ ↑ [55] Avian v-Fps Non-receptor Tyr EAEIYEAIE No O ↓ ↑ [56] Bovine PKA Non-receptor Ser/Thr LRRASLG ↓ ↓ ↑ [50] Human CDK2 Non-receptor Ser/Thr Histone H1 ↓ ↓ ↑ [57] Human Bruton’s tyrosine kinase Non-receptor Tyr Poly-E4 Y ↑ ? ↑ [58] Human EGFR (ErbB1) Receptor Tyr Peptide A ? ? ↑ [59] Human ErbB2 Receptor Tyr Peptide B ? ? ↑ [59] Human ErbB3 Receptor Tyr Peptide C ? ? ↑ [59] Human Yes Receptor Tyr Poly-E4 Y ? ? ↑ [60] Human VEGFR2 Receptor Tyr Poly-E4 Y ? ? ↑ [61]
a LRRK2, leucine-rich repeat kinase-2; IRAK-4, interleukin-1 receptor associated kinase-4.
b Peptide I, RLGRDKYKTLRQIRQ; Peptide II, PKTPKKAKKL; Peptide III, KKARFSRFAGSSPSQSSMVAR; Peptide A (biotin-(aminohexanoate)-EEEEYFELVAKKK-CONH2 ); Peptide B (biotin-(aminohexanoate)-GGMEDIYFEFMGGKKK-CONH2 ); Peptide C (biotin-(aminohexanoate)-RAHEEIYHFFFAKKK-CONH2 ).

R. Roskoski Jr. / Pharmacological Research xxx (2015) xxx–xxx 11

Fig. 6. Proposed protein kinase catalytic cycle including Mg2+(1), Mg2+ (2)-ATP, pro- tein substrate, and Mg2+ (2)-ADP.
Source: Adapted from Bastidas et al. [67].

In contrast to the above studies, Gerlits et al. reported that PKA can mediate the phosphorylation of a high afﬁnity peptide (SP20) in the absence of a divalent cation [68]. This study demonstrated that divalent metals greatly enhance catalytic turnover. Moreover, in the absence of Mg2+ or other metal, these investigators showed that PKA mediates but a single turnover from ATP to the high- afﬁnity peptide. In another study, Mukherjee et al. reported that Ca2+/calmodulin-activated Ser-Thr kinase (CASK) functions with- out a divalent cation [69]. This enzyme, which lacks the critical D of DFG that binds to Mg2+, was thought to be an inactive pseudok- inase. However, CASK exhibits catalytic activity and, surprisingly, catalysis is actually inhibited by Mg2+, Mn2+, or Ca2+.

Targeting the Mg2+binding sites
The elucidation of the role of two Mg2+ ions suggests other strategies for the development of Src inhibitory drugs. The design of ligands that bind to the ATP-binding pocket with an extension that interacts with either the metal-binding asparagine within the catalytic loop or the metal-binding aspartate at the beginning of the activation segment promises to yield new types of protein kinase inhibitor. Along these lines, Peng et al. developed EGFR inhibitors that form a salt bridge with Asp831 of its DFG-motif [70], which may interfere with the binding of Mg2+(1) to the enzyme. It remains to be established whether this strategy will have general applicability.

Src signaling and cancer

Src is a non-receptor protein tyrosine kinase that participates in numerous signaling pathways [4]. Src interacts with several

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Fig. 7. Src signaling pathways. FAK, focal adhesion kinase; MAPKs, mitogen-activated protein kinases.

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Insulin-like growth factors (IGF-1 and -2) participate in several processes including cell division, growth, survival, angiogenesis, wound healing, and embryonic development [76,77]. These growth factors bind to the insulin-like growth factor receptor (IGFR) and to the insulin receptor; they bind to IGFR with higher afﬁnity than they bind to the insulin receptor. IGFR dysregulation is implicated in a variety of human cancers including breast, colorectal, and prostate cancers, NSCLC, and sarcomas. IGFR dysregulation is related in part to increased production of IGF-1 and -2.
In humans, 22 members of the ﬁbroblast growth factor (FGF) family and four protein-tyrosine kinase receptors (FGFR1–4) have been identiﬁed. FGFR signaling regulates cell growth and division in many cell types in addition to ﬁbroblasts. FGFR signaling is involved in angiogenesis, wound healing, and embryonic development. FGF family signaling is implicated in hepatocellular carcinoma, melanoma, lung, breast, bladder, endometrial, head and neck, and prostate cancers [78]. Point mutations and gene ampliﬁca- tion/overexpression of members of the FGFR family are responsible for dysregulation and oncogenesis. Point mutations have been described in 50–60% of urothelial carcinomas and gene ampliﬁca- tion has been described in 10% of breast carcinomas.
Besides protein-tyrosine kinases, Src-family kinases are con-
trolled by integrin receptors, G-protein coupled receptors, antigen- and Fc-coupled receptors, cytokine receptors, and steroid hormone receptors [4]. Src participates in cell migration and motility by interacting with integrins, E-cadherin, and focal adhesion kinase (Fig. 7) [30]. Src participates in pathways regulating cell survival, proliferation, and regulation of gene expression [4]. The enzyme also plays an essential role in bone formation and remodeling and may play a role in breast, prostate, and lung cancer metastasis to the skeleton.

Therapeutic small molecule Src inhibitors

Src as a drug target

The role of v-Src in oncogenesis eventually led to the dis- covery of the Src proto-oncogene and then to the discovery

of all of the other members of the Src family of protein kinases. Src drug discovery has been aimed at the role of Src in oncogenesis. Indeed, most of the FDA-approved small molecule inhibitors of protein kinases are directed toward neoplastic dis- eases (www.brimr.org/PKI/PKIs.htm). Unlike BRAF, EGFR, or ALK mutants or BCR-Abl fusion proteins, Src is not a primary driver of tumorigenesis, but rather it is a participant in many pathways pro- moting cell division and survival. Moreover, Src mutants in tumors are very rare. Thus, it is unlikely that anti-Src monotherapy will be efﬁcacious in the treatment of cancers. Since Src is a participant in many aspects of cell division, invasion, migration and survival, Src inhibition may play an important auxiliary role in various cancer treatments as described in the next section.

Src inhibitors that are FDA-approved or in clinical trials

As noted above, Src and Src family kinases have been implicated in the neoplastic process for three decades and extensive work on the development of Src inhibitors has been performed [79]. Src is downstream from such oncogenic drivers as EGFR, ErbB2, and BCR-Abl. Signals downstream from these oncogenic drivers include the Ras/Raf/ERK cell division pathway and the phosphatidylinositol 3-kinase and protein kinase B (Akt) cell survival pathway [80], which are pathways that involve Src. Thus far there appears to be no prognostic biomarkers related to Src activity that can be used for patient selection in clinical trials. Moreover, Src-speciﬁc kinase inhibitors have not made their way into the clinic.
Four orally effective Src/multikinase inhibitors are FDA- approved for the treatment of various malignancies (Table 4). Bosutinib is a BCR-Abl, Src, Lyn, Hck, Kit, and PDGFR inhibitor that is approved for the treatment of Ph+ (i) CML and (ii) ALL (Fig. 8). This drug is currently in clinical trials for the treatment of breast cancer and glioblastoma. Dasatinib is an inhibitor of BCR-Abl, Src, Lck, Fyn, Yes, PDGFR, and other kinases that is approved for the treatment
of CML. This drug is undergoing numerous clinical trials for vari- ous solid tumors and for ALL. Ponatinib is an inhibitor of BCR-Abl, PDGFR, VEGFR, Src family and other kinases that is approved for

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R. Roskoski Jr. / Pharmacological Research xxx (2015) xxx–xxx 13

Table 4
Selected orally effective Src/multikinase small molecule inhibitors.

Drug Known targets PubChem CIDa Clinical indicationsb References
Bosutinib BCR-Abl, Src, Lyn, Hck, Kit, PDGFR 5328940 Ph+ CMLb , Ph+ ALLb, breast cancer, [81–83]
glioblastoma
Dasatinib BCR-Abl, Src, Fyn, Yes, Lck, Arg, Kit, 3062316 Ph+ CMLb , Ph+ ALL, breast, colorectal, [81,84]
EphA2, EGFR, PDGFRþ endometrial, head and neck, ovarian, and
small cell lung cancers, glioblastoma,
melanoma, and NSCLC
Ponatinib BCR-Abl, Src family kinases, VEGFR, 24826799 Ph+ CMLb , Ph+ ALLb, endometrial, GIST, [81,85]
PDGFR, FGFR, Eph, Kit, RET, Tie2, hepatic biliary, small cell lung, and thyroid
Flt3 cancers
Vandetanib RET, Src family kinases, EGFR, 3081361 Medullary thyroid cancerb, breast, head [86–88]
VEGFRs, Brk, Tie2, EphRs and neck, kidney cancers, NSCLC, and
several other solid tumors
Saracatinib (AZD0530) Src, BCR-Abl 10302451 Colorectal, gastric, ovarian, small cell lung [81,89]
cancers, NSCLC, and metastatic
osteosarcoma in lung
AZD0424 Src, BCR-Abl None Early clinical trials for numerous solid http://www.clinicaltrials.gov/
tumors
a The PubChem CID (chemical identiﬁcation no.) from the National Library of Medicine (http://www.ncbi.nlm.nih.gov/pubmed) provides the chemical structure, molecular weight, number of hydrogen-bond donors/acceptors, and bibliographic references.
b Indication approved by FDA, otherwise in clinical trials.

Fig. 8. Structures of selected Src/multikinase inhibitors approved by the FDA or in clinical trials.

976 the treatment of CML and ALL. It too is undergoing clinical trials for their approval for the treatment of Ph+ CML. The data in Table 4 995
977 several solid tumors. indicate that these drugs inhibit more than their initial targets, 996
978 Vandetanib is an inhibitor or EGFR, VEGFR, RET, Src family and and this property is shared by most of the FDA-approved kinase 997
979 other kinases that is approved for the treatment of medullary thy- inhibitors (www.brimr.org/PKI/PKIs.htm). Whether these drugs are 998
980 roid carcinoma, and it is in clinical trials for numerous solid tumors clinically effective for the treatment of various solid tumors and 999
981 (Table 4). With the exception of vandetanib, the currently FDA- whether such effectiveness is related to primarily to Src inhibi- 1000
982 approved disease targets of these drugs are hematologic in nature tion or to the inhibition of other protein kinases remains to be 1001
983 and not directed against solid tumors. Saracatinib (AZD0530) is determined. 1002
984 a Src and BCR-Abl inhibitor that is undergoing clinical trials for
985 colorectal, gastric, ovarian, small cell lung cancers, NSCLC, and
986 metastatic osteosarcoma in lung (www.clinicaltrials.gov). A related The ATP-binding pocket of Src 1003
987 drug (AZD0424) is in stage I clinical trials for numerous solid The glycine-rich loop occurs universally in protein kinases and 1004
988 tumors. KX01, KX2-391, XL228, XL99, and XLI-999 are Src inhibitors consists of a canonical GxGx8G sequence where 8 refers to a 1005
989 that were in clinical trials against various disorders, but they have hydrophobic residue. In Src this sequence consists of GQGCFG 1006
990 a low likelihood of advancing in the clinic. (Table 2). The glycine-rich loop, which forms a lid above the ATP 1007
991 Bosutinib [90], dasatinib [91], ponatinib [92], saracatinib [93], phosphates, is characteristically one of the most mobile portions of 1008
992 and AZD0424 [94] were initially developed as Src/Abl inhibitors the protein kinase domain. This mobility may be due to the require- 1009
993 and vandetanib [95] was initially developed as a VEGFR2 inhibitor. ment that the enzyme binds ATP and then releases ADP following 1010
994 Inhibition of Abl by bosutinib, dasatinib, and ponatinib accounts for catalysis. The penultimate phenylalanine and third glycine of the 1011

14 R. Roskoski Jr. / Pharmacological Research xxx (2015) xxx–xxx

Fig. 9. (A) Binding of ATP-μ-S to active chicken Src (PDB ID: 3DQW). (B) Binding of dasatinib to active human Src (PDB ID: 3QLG). (C) Superposition of ATP-μ-S and dasatinib bound to Src. (D) Superposition of dasatinib (PDB ID: 3QLG) and bosu- tinib (PDB ID: 4MXO) bound to Src. Ado, adenosine; AS, activation segment; H8,

ATP-competitive Src inhibitors

The two main classes of reversible ATP-competitive protein kinase inhibitors are named type I and type II [98]. Type I inhibitors bind to the DFG-Asp in enzyme conformation and the type II inhibitors bind to the DGF-Asp out conformation. The X-ray crys- tallographic structures of dasatinib [99] and bosutinib bound to Src demonstrate that these are type I inhibitors. The structures of ponatinib, vandetanib or saracatinib bound to Src have not been reported (www.pdb.org). The interactions of dasatinib and bosu- tinib with Src are similar so that only dasatinib is considered in detail here.
Most, if not all, ATP-competitive protein kinase inhibitors inter- act with the peptide backbone of hinge residues, and dasatinib and bosutinib are not exceptions. The thiazole nitrogen of dasatinib forms a hydrogen bond with Met344 of the hinge (Fig. 9B). ATP- competitive protein kinase inhibitors generally interact with the nearby C-spine residues. In the case of Src, dasatinib interacts with Ala296 and Leu396. It also forms hydrophobic contacts with Tyr343, Thr341, Ile339, and Val284. Thr341 is the gatekeeper residue and Ile339 is the Sh3 shell residue. Dasatinib also extends to the aC- helix and makes hydrophobic contacts with Met317, which is RS3 of the regulatory spine. Bosutinib has hydrophobic interactions with all of these residues with the exception of Ile336 (PDB ID: 3QLG) (not shown).
Adenine interacts with residues within the þ1, þ2, and þ3- strand in the N-lobe while most ATP-competitive inhibitors including dasatinib and bosutinib extend into a region called hydrophobic pocket II or the back pocket [98] that continues past the þ5 and þ4-strands to the aC-helix. Both dasatinib and bosutinib bind to the active form of Src with the aC-helix in conformation (Lys298 and Glu313 are kissing) and with the activation segment in its open conformation. The superposition of bound ATP-μ-S and dasatinib depicts the extension of the drug that extends into the hydrophobic pocket (Fig. 9C). Moreover, the superposition of dasa- tinib and bosutinib bound to Src illustrates their binding similarities (Fig. 9D).

Epilogue

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