Chapter 24. SH2 and SH3 Domains: Choreographers of Multiple Signaling Pathways

Chapter 24.

SH2 and SH3 Domain.: Choreographer. Signaling Pathway.

of Multiple

Martyn C. Botfield and Jeremy Green ARlAD Pharmaceuticals, Cambridge. MA 02139

introduction - Cellular signal transduction is a deceptively simple concept: information

is gleaned from the external environment and the appropriate preprogrammed cellular response is executed. Not surprisingly, the molecular r e a l i of these events belies the conceptual simplicity. Individual cells must respond correctly to dozehs of simultaneous stimuli and integratethe multiple signal cascades. Rather than being a simple performance by one or two principals and a small chorus, reality within the cell is more akin to several intricate productions being performedon the same stage at the same time. To complicate matters further, many of the principal players are shared between the multiple productions and must flawlessly enter and exit the various performances without impairing the movement of the other participants. How then is chaos averted? To a large degree signal transduction pathways are choreographed by modular SH2 and SH3 domains which mediate highly specific protein:protein interactions. SH2 (Src homology 2) domains are modules of -100 amino acids that specifically bind phosphotyrosinecontaining proteins and peptides (1,2). SH3 (Src homology 3) domains are modules of -60 amino acids that bind to proline-rich sequences (3,4). A list of selected therapeutic targets possessing SH2 or SH3 domains is given in Table 1. The design of specific antagonists to these domains holds the promise of targeted treatment of a broad range of pathologies. TAB1 F 1.

Selected list of SH2- and SHB-containing therapeutic targets and their associated pathologies.

PATHOLOGY

TARGET

DOMAIN STRUCTURE

REFERENCES

AIDS

k%

SH3-SH2-klm SH3-SH2-kl~se

5

Akrgymd Amthrm

Anemla

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SH2-SHPphosPhatase

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ZAP

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12-14

Breast

Gtb2 -7 Src

sH3-sH2-sH3

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SHSSH2-SH2

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PH-SH2 SHS-SM-klm

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SH2 SHSSH2-SH3 SH2SH3-SH2-PW

26

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SHSSH2-SH3 sH2-sH3.sH3

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Erylhrokukemlu

Shc

SH2

29

Inllamnutory D l w u o

STATs p47-PhOx p67-phox

DNA-blndlng-SHSSH2 SHSSH3

s 3 2

SHSSH3

33-35 33-35

PKSHSSH~-~~MSO

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Gap

CML a d ALL

ANNUAL REPORT8 IN MEDICINAL CHEMLSTRYJO

2x7

PH-SHSSH2-klnase

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SH3-SH2-klm

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Beotlon V-Toplos ln Biology

h e . Ed

Schematized signal transduction pathways.

KING THROUGH MEtvlBRANE RECFPTORS The role of SH2 and SH3 domains in signal transduction has been extensively reviewed (1,2,40-43). In growth factor, cytokine and antigen signaling, occupancy of a receptor by agonist results in receptor dimerization and the phosphorylation of regulatory tyrosines on the cytoplasmic surface (44). Phosphorylation is catalyzed by Wnases that are a part of the receptor (receptor tyrosine kinases) or recruited to the receptor from the cytoplasm (non-receptor tyrosine kinases). The resulting phosphotyrosines permit binding of specific SH2containing proteins and initiate a cascade of sequential protein interactions (Figure 1). Recruited molecules may themselves be tyrosine kinases and catalyze additional cycles of phosphorylation and SH2 recruitment (45); tyrosine phosphatases that terminate SH2-mediated associations (46,47); kinases or phosphatases that activate or inactivate previously bound enzymes (47); enzymes that generate lipid-derived second messengers (48); SHWSH3 adapter proteins that act as docking stations for additional signaling molecules (49); or proteins that ultimately translocate to the nucleus and regulate transcription (30-32,50). Thus a single event extracellular binding of agonist can be coupledto a broad range of cellular responses through a series of SH2- and SH3mediated events. These cellular responses include proliferation and differentiation (28,32,51-54), transcription (30-32,50), programmed cell death (55-57), adhesion, cytoskeletal rearrangement and chemotaxis (58,59), exo- and endocytosis (60,61), and assembly and activation of multi-subunit enzyme complexes (33-35).

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SH2 domains are small independently folded protein modules that bind to phosphotyrosine and permit phosphorylation dependent protein:protein interactions. The high affinity and specificity of the interaction permits a small numbers of molecules to survey and report the information state of the cell with a low risk of initiating false signals through random collision with other signaling molecules.

SH2 and 5H3 Domsins

Chap.24

Botfleld. Oreen

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-

The three dimensional structure of a variety of ligated and unligated SH2 domains have been determined: src(62-64), Lck (65), PLCyl (66), Abl (67,68), P13K (69), SH-PTP2 (70), and the tandem SH2 domains of ZAP-70 (71). Together they define a common structural fold, i.e. a central anti-parallel P-sheet bounded on either side by an a-helix. The two binding sites -- one on either side of the P-sheet are formed by close apposition of the helices to the sheet. The minimal phosphopeptide ligand is four amino acids (pYXXX) with a preference for a hydrophobic residue in the pY+3 position (72). Bound phosphopeptide is in an extended conformation that straddles the SH2 domain surface and is largely solvent exposed. The bulk of the interactions with the peptide involve phosphotyrosine and the pY+3 residue. Thus binding of phosphopeptide has been described as a 'two pronged plug engaging a two-holed socket" (73). Only a small contribution is made by the pY+1 residue and little or no contact is made by the pY+2 residues or sites outside of the core motif. These structural predictions agree well with in vifro binding data (63,74). The phosphotyrosine binding cleft is formed by three strands of the P-sheet (PB, PC and PD), the loop between PB and PC (BC loop) and aA2 of the A helix (62-64). The PB strand is the site of the highly conserved FLVBES sequence (75). The central arginine of this sequence (Arg-178 in Src) is the only invariant residue and forms two hydrogen bonds with phosphate oxygens of the phosphotyrosine. Mutation of Arg-178 abolishes binding (74). It is likely that the position of this residue deep within the binding pocket places it beyond the reach of phosphoserine and phosphothreonine and thus explains the absence of demonstrable binding to peptides containing these residues. The hydrophobic pY+3 binding pocket is formed by two strands of the P-sheet (PD and BE) and the loops between PUPF and aB/PG. The precise formation and sidechain composition of this pocket is more variable than that of the phosphotyrosine pocket and generates differences in both affinity and specificity (76,77). This is most notable in PLCyl and SH-PTP2 (Syp) where the pocket is reshaped to form an extended groove running across the ligand-binding surface (66,70). Molecular dynamic studies using nuclear magnetic resonance spectroscopy indicates that ligand binding induces little structural rearrangement other than side chain position within the binding clefts. Only the BC loop shifts towards a tighter packing with the phosphopeptide and protein core (78).

--

Afflnitv

-

The affinity of single SH2 domains for phosphoproteins and phosphopeptides derived from physiological targets range significantly. Using a variety of techniques, estimates from 10-5 to 10-9 M have been reported (72,76,7981). Isothermal titration calorimetry suggests that the average change in standard free energy (AGO) for the interaction between a single SH2 domain and a monophosphorylated ligand is approximately -8.5 kcalmol-1 (81). Essentially no binding is detected when phosphotyrosine is replaced by tyrosine. Dissociation rates are very rapid (Kdi,>O.l sec-1 yielding t ,+0.2 minutes) for single SH2 domains (79,82,83). Tandem domains (SH2-SH2) have higher affinity (5lO-QM) and slower dissociation rates (hiss lOO minutes) for tandem binding sites suggesting that specificity, affinity and signal duration can all be amplified by the multimerization of binding domains. Tandem SH2 domains are found in a variety of proteins including Syk, ZAP-70, PLCy, the .p85 subunit of P13K, SH-PTP1 and SH-PTP2.

Specificity - In vitro studies suggest that for isolated SH2 domains, the difference between the binding affinities of specific and non-specific phosphopeptides ranges from 10-1000 fold (63,76,83-85). Using degenerate phosphopeptide libraries, Cantley and colleagues determined the preferred binding sequences for Src, Fyn, Lck, Fgr, Abl, Grb2, Drk, Csk, Vav, SHC, fpdfes, Crk, Nck, Sem5, SH-PTP1, SHPTP2, Syk, PLCyl and the p85 subunit of P13K (72,86). The SH2 domains from p85 selected pYMXM or pYVXM, the exact sequences that have been identified as the p85-binding sites on the PDGF receptor, colony-stimulating factor 1 receptor and c-kit. All members of the Src family that where examined (Src, Fyn,

Beotlon V-Topics In Blologv

Lee. Ed

Fiaure 2 SH3 Class I (ZPYPPW) versus Class II (YPPYPPZ,) ligand binding. P = Proline, Y = hydrophobic amino acid, Z = SH3specific amino acid. See text for complete description. Lyn, Lck and Fgr) selected pYEEl and defined a general consensus -of pYEEY, where Y represents hydrophobic residues. Other members exhibited varying degrees of preference, usually with stronger selection for residues at the pY+3 and less selection at pY+1 and Y+2, in concurrence with prediction from structure. An exception to this rule were the SH2 domains from Sem5iGrbUDrk which exhibited a marked preference for Asn at the pY+2 position and less preference at the pY+1 and pY+3 pockets. It is anticipated that expanding the set of structural monomers beyond the 20 naturally occurring amino acids will permit the synthesis of exquisitely selective SH2 antagonists. SH3 DOMAINS SH3 (Src homology 3) domains are independently folded protein modules of 55-70 amino acids that selectively bind proline-rich protein sequences (3,4). Although generally of lower affinity than SH2-mediated interactions, SH3 interactions are essential for multiple signaling cascades. w t u r e - A variety of ligated and unligated SH3 structures have been reported including Src (87-89), P13K (90-92), PLCy (93), GAP (94), Fyn (95), Csk (96), Lck (65), Abl (97), GRB2 (98) and u-spectrin (88). Although there is variability, the basic fold is composed of eight Fstrands arranged in two, three-member anti-parallel p-sheets (PI and pll) that form a compact core and a third, two-strand p-sheet @Ill; ref. 98). The binding "platform" is formed by residues of fill and pill plus two negativelycharged hypervariable loops and is subdivided into three binding sites: a polar pocket and two hydrophobic pockets. Details of ligand binding are discussed below.

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k & L Y mmlhg Peptides bind as left-handedpolyproline type II helices (89) in one of two orientations (Figure 2). Class I peptides bind with their aminotermini making contact with the polar pocket and carboxy-terminal residues intercalating the hydrophobic pockets. Class II peptides bind in the reverse orientation, i.e. with the carboxy-terminus making contact with the polar pocket and amino-terminal residues intercalating the hydrophobic sites. In both cases the same binding sites are occupied; only the orientation is reversed (89). Class I vmus Class II orientation can be deduced directly from the peptide sequence (99). Class I ligands have the general form LPVPPYP and Class II ligands VPPYPPZ where Y is any hydrophobic amino acid and b is an SH3-domainspecific residue (commonly arginine). When oriented in the binding platform, Z makes contact with the polar binding pocket and the two Y-Pro motifs occupy the two hydrophobic pockets. The proline residues that connect the three submotifs, Le. ZPYPPY P (Class I) and Y PEY PPZ (Class Il), form the third, non-contacting ridge of the proline helix and stabilize helix integrity. Class I versus Class II orientation is dictated by several features: the position of the Lresidue relative to G

chap. 24

8H2 and 8H3 Domsins

Botfleld. G r e e n

231

the Y P motifs; the position of the y and 6 methylenes of proline within the pocket; and the uniquely compact nature of the Y-Pro dipeptide. In a Y-Pro sequence, the C a 4 p bond of Y is separated from the N-Cg bond of proline by two backbone bonds; in a Pro-Y sequence, this separation is three backbone bonds. Thus a Pro-Y sequence has a composite hydrophobic surface that is significantly larger than that of an Y-Pro sequence and can not be accommodated by the binding site (89). Details of these features and their precise role in determining orientation are reviewed elsewhere (89,99,100).

Afflnitv

-

Estimates of the affinity of single SH3 domains for intact proteins or peptides derived from physiological targets range from >lO-3 to lO-7M (25,89,92,98, 101-104) and are thus generally weaker than SH2/phosphotyrosine interactions. These modest affinities can be enhanced by multimerization. For example, the affinity of the adapter protein Grb2 (SH3-SH2-SH3) has a 6 - 2 5 nM for a substrate that permits simultaneous interaction with both SH3 domains (25). No inherent difference in Class I versus Class II affinity has been reported. Soecificu - SH3 specificity has been explored using phage display (102,105,106) and random peptide libraries (92,101). Core motif peptides are relatively nonselective and bind to a broad range of SH3 domains. For example, the optimum Class I core for Src is BPLPPLP (102,105,106). This sequence also binds to the SH3 domains of Lyn, Fyn, Yes, and P13K (102). It does not, however, bind to the Abl SH3 domain (102). This selectivity is imparted by the amino-terminal arginine and marks the first determinant of specificity, i.e. the first (ZPYPPYP) or last (YPPYPPZ) residue of Class I or Class I1cores, respectively. SH3 domains with an aspartic or glutamic acid in a conserved position of the polar binding pocket (Asp99 in Src) all bind peptides where Z is arginine. SH3 domains that have a nonacidic amino acid at this position, e.g. Abl, fail to bind arginine peptides and select for alternative residues. Additional specificity is derived from as many as five residues either side of the core, i.e. XXXXZPYPPYPXXXX or XXXXYPPY PPZXXXX (102). Flanking sequences specific for the SH3 domains of Src, Fyn, Lyn, Yes P13K and Abl have all been reported (102).

Human pathologies correlated with dysfunction of SH2 and SH3 domains are listed in Table 2. The precise molecular contributions of these domains in precipitating disease have been identified in several cases. The following examples are chosen to illustrate both the complexity of the cellular signaling pathways and the diversity of roles fulfilled by SH2 and SH3 domains.

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c Mveloaenous Lwkemta (CMU and Acute LyOlphoblastic Leukemia (A1 I ) A reciprocal translocation between chromosomes 9 and 22 (the so called "Philadelphia chromosome") has been identified in many cases of CML and ALL (Ph+ CML and Ph+ ALL). This translocation results in the juxtaposition of the bcr gene and the abl proto-oncogene (107,116) and results in expression of 185 kDa or 210 kDa chimeric Bcr-Abl proteins correlated with ALL and CML, respectively (117,118). In both cases, the first 26 amino acids of the proto-oncbgene c-Abl are replaced by the first 426 or 927 (or 902) amino acids of Bcr (107,117,118). The resulting chimeras localize to the cytoplasm and contain the SH3, SH2 and tyrosine kinase domains of c-abl. The presence of the Bcr sequence deregulates the kinase, an essential requirement for transformation (119,120). Bcr-Abl forms a physical complex with a variety of signaling molecules including Grb2 (27,l lo), SK PTP2 (121), and CrkL (122). Grb2 is a 25 kDa adapter protein of the form SH3SH2-SH3 sequence. The SH2 domain of Grb2 recognizes and binds to the bcrencoded Y177 tyrosine autophosphorylation site in both the p185 and p210 BcrAbl proteins. Grb2 in turn associates with mSOS (a Ras guanine nucleotide exchange protein) through a Grb2-SH3/mSOS-poly-proline interaction and

Lee, Ed

Bemion V-Topioe In Biology

IAELE-2

Selected human pathologies correlated with dysfunction of SH2and SH3-containing proteins.

PATHOLOGY

PROTEIN

DOMAIN STRUCTURE

REFERENCES

Chronic Myelogenous Leukemia

BcrIAbi Grb2 CM

SHS-SH2Wnase SH3-SH2-SH3 SW-SH3-SH3

X-Linked Agammaglobulinemia

Btk

PH-SH3-SHP-kinase

111.1 12

Myelodysplastlc Syndrome

TeC

PH-SH3-SH2-kinase

37

Chronic Granulomatous Disease

p47-phox p67-phox

SH3-SH3 SH3-SH3

35 35

Severe Combined immunodeficiency

ZAP-70

SHP-SHP-kinase

113,114

Faclogenltal Dyspiasia

FGD1

SH3-binding site

115

107-109 27,110 28

(activates the Ras pathway leading to deregulated mitogenic signals (27,110). Mutation of Y 177 to phenylalanine (Y177F) abolishes GRB-2 binding and abrogates Bcr-Abl-induced Ras activation and transformation (27). Conversely, point mutations that severely impair the ability of the SH2 domain to bind phosphotyrosine R552L in pl85) or removes the major tyrosine autophosphorylation in the kinase domain (Y813F in p185), also impair transformation by Bcr-Abl without effecting Grb2 binding (123). These results suggest that Grb2 binding is necessary, but not sufficient for transformation. The second factor may be CrkL, a 39 kDa adapter protein of the form SH2-SH3-SH3. In neutrophils from normal patients, CrkL is unphosphorylated. In neutrophils from Ph+ CML patients (28,124) or bcr-abl transformed cell lines (122), CrkL is highly and constitutively tyrosine phosphorylated. Treatment of normal neutrophils with a variety of cytokines and agonists fails to induce CrkL phosphotylation suggesting that this is not part of a normal signaling pathway (124). Furthermore, CrkL forms a physical complex with Bcr-Abl in transformed cells and is readily tyrosine phosphorylated by the Bcr-Abl and c-Abl kinases in vitro (122). Together these results implicate CrkL as a second potential oncogenic mediator of Bcr-Abl. Chronic G w l o m a t o u s Disease (CGU - CGD is characterized by a failure of neutrophils to generate microbicidal oxidants (e.g. superoxide) and leaves CGD patients highly susceptible to opportunistic infections (35). The enzyme responsible, NADPH oxidase, is composed of four proteins: p22- phox, p47-phox, p67-phox and gp91-phox. Activation of NADPH oxidase is regulated through assembly, i.e. upon stimulation of the phagocyte, p47-phox and p67-phox are translocated from the cytosol to the membrane where they associate with the membrane-bound p22-phoxlgp91-phoxlheme complex and activate superoxide generation. This process relies on a series of SH3-mediated events that begins with the dissociation of an intramolecular proline-rich binding sequence from the p47-phox SH3-domain that unmasks the SH3 domain (34). Following unmasking, both the SH3 and proline-rich sequences become available to participate in other intermolecular interactions. Unmasked p47-phox then associates with p67- phox through SH3-mediated interactions to form a p47lp67 heterodimer (34) which in turn associates with the membrane-bound components through one or both SH3 domains of p47-phox and proline-rich sequences of p22-phox (34). Mutation of Pro-156 to glutamine in the p22-phox disrupts one proline-rich sequence and abolishes binding (33,34). An identical mutation has been found in a patient with CGD (125), suggesting that it represents a physiological binding sequence. Synthetic peptides corresponding to proline-rich SHSbinding sites of p22-phox or p47-phox effectively block in vitro oxidase assembly and activation (33,34,126).

8H2 and 8H3 Domains

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233

The design of SH2 inhibitors presents novel challenges. Paramount among these is cell permeability and phosphatase resistance. Although few compounds have been reported in the literature, several phosphatase resistant mimics and cellpermeable phosphate "pro-drugs" deserve note.

. .

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PhosDhoprosine Mlmlcs Phosphotyrosine (1)can most easily be rendered resistant to phosphatases by replacetnent of the tyrosine oxygen with a CH, as in 8. Numerous syntheses, both racemic (127) and asymmetric (128), have been reported. Peptides containing 3 as a replacement for phosphotyrosine show a minimum 6-fold weaker binding depending on the SH2 domain examined (63,129,130). Crystal structures of the Lck SH2 domain in complex with two separate peptides prepared with 5 have been solved (131). The conformation of the SH2 domain is largely unchanged from the phosphotyrosine complex, with the notable exception of a perturbation in the BC loop ("phosphotyrosine-binding"loop) that connects strands PB and PC. The observed position of the BC loop suggests the loss or absence of up to three hydrogen bonds between the loop and the phosphonate. It has been rationalized that the decreased affinity of this analog may be a result of either the lower acidity of this residue (pK,2 = 7.1) relative to phosphotyrosine (pK,2 = 5.7), or the loss of hydrogen bonding between the phosphate oxygen SH2 domain, or a combination of both (130). To test these hypotheses, peptides were prepared with phosphonate analogs bearing electron withdrawing substituents a to phosphorus in order to increase the acidity of the phosphonate. Inclusion of a single fluorine atom @) increased affinity 2-fold, while a second fluorine (2) re-established a level of binding equal to phosphotyrosine (1). The utility of peptides prepared with 2 has been demonstrated in T-cells. Peptides corresponding to the third immunoreceptor tyrosine-based activation motif (ITAM) of the T-cell receptor {-chain were able to selectively inhibit the association of the Tcell receptor with the tyrosine kinase ZAP-70 in permeabilized T-cells (14). 2containing peptides also effectively block SH-PTP2 mediated mitogenic signaling in rat fibroblasts (132). In both studies, the equivalent phosphotyrosine peptides showed no efficacy, presumably due to removal of the labile phosphate via cellular phosphatases. relative affinity

1 2

eR $ 4

5 6

a7

H2N

R=OP03H2 R=CF2P03H2 R=OPS02H2 R=CHFP03H2 R=CH2P03H2 R = CHOHPO3H2 R = CHOHP03H2 R=CH2P02H2

9 1p R=CH2S03H R=CH2CH2COCHO

0

11 li!

a

14

fi

R = CH2CH2COCOCH3 R=CH2COOH R = CH2CONHOH R=NH2 R=N02 R=OH

1 1 2 3 6 20 25 40

300 370 520 940 >loo0 >loo0 >loo0 >loo0

In extensive studies of the binding preferences of the Src SH2 domain for peptidebased ligands (63,133,134), a number of potential phosphotyrosine mimics were assayed against Src SH3-SH2 with variable effectiveness (see table associated with I-=). Although essentially inactive, compounds 1p and 11 represent a novel

attempt to "trap" the arginine residues that participate in phosphotyrosine binding. Several non-phosphorus mimics (a-I.2)were also examined with II being the only candidate exhibiting activity (135). Although peptides incorporating l .2 showed no affinity for Src and Grb2, peptides specific for P13K showed measurable, albeit 100fold reduced, affinity for the p85 SH2 domains (63). Furthermore, binding of 1z to the N-terminal SH2 of the phosphatase SH-PTPP was equal to that observed with the corresponding &containing peptide. Cellular studies using peptides prepared with will be valuable in assessing the potentials of this promising phosphotyrosine mimic.

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tdPhosphonate Pro-druw Should the challenge of creating a cell permeable phosphotyrosine mimic prove elusive, the alternative strategy of neutralizing the dianionic phosphate in the form of a pro-drug may be viable. The use of phosphate diesters have previously been applied in other areas where masking of phosphate is required, such as nucleotide anti-virals (136). One limitation of the diester protecting group approach is the reduced susceptibility of the anionic protecting group monoester to hydrolytic enzymes following cleavage of the first ester group from the neutral molecule. This can be circumvented by removing the site of hydrolysis far from the phosphorus atom, such as in the application of di(4acetoxybenzyl) phosphonates (137). A recent novel approach is presented in a model "masked" phosphate, M. Through the action of cellular esterases, the labile pivaloyl ester is converted to an unstable hemiacetal phosphate triester, which decomposes to reveal the phosphate (138). It will be interesting to see how these and others compounds evolve.

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(1=4.

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10. 11.

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N. Ishii, H. Asao, Y. Kimura, T. Takeshita, M. Nakamura, S. Tsuchiya, T. Konno, M. Meede, T. 1310 (1994). Uchiiamaand K Sugarnura, J. Irnmunol. R.L. Wange, N. Isakov, T.J. Burke, A. Otaka, P.P. Roller, J.D. Watts, R. Aebersdd and L.E. Samelson, J. Bid. Chern. 944 (1995). P.W.Janes, R.J. Daly,A. deFazi0 and R.L. Sutherland,Oncogenes, 3601 (1994). R.J. Daly, M.D. Binder and R.L. Suthefiand, Oncogene g, 2723 (1994).

8H2 and 8H3 Domain8

Chap. 24

17. 18. 19.

a. 21. 22.

P

24.

25. 26, 27.

28. 29. 30. 31. 32 33. 34.

36. 36. 37. 38. 39. 40. 41. 42 43, 44. 45. 46. 47. 48. 49. 50. 51.

5? 53. 54. 55. 56.

n. 58.

59.

60.

61.

M

63.

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