Essential role of PSM/SH2-B variants in insulin receptor catalytic activation and the resulting cellular responses

The positive regulatory role of PSM/SH2-B downstream of various mitogenic receptor tyrosine kinases or gene disruption experiments in mice support a role of PSM in the regulation of insulin action. Here, four alternative PSM splice variants and individual functional domains were compared for their role in the regulation of specific metabolic insulin responses. We found that individual PSM variants in 3T3-L1 adipocytes potentiated insulin-mediated glucose and amino acid transport, glycogenesis, lipogenesis, and key components in the metabolic insulin response including p70 S6 kinase, glycogen synthase, glycogen synthase kinase 3 (GSK3), Akt, Cbl, and IRS-1. Highest activity was consistently observed for PSM alpha, followed by beta, delta, and gamma with decreasing activity. In contrast, dominant-negative peptide mimetics of the PSM Pro-rich, pleckstrin homology (PH), or src homology 2 (SH2) domains inhibited any tested insulin response. Potentiation of the insulin response originated at the insulin receptor (IR) kinase level by PSM variant-specific regulation of the Km (ATP) whereas the Vmax remained unaffected. IR catalytic activation was inhibited by peptide mimetics of the PSM SH2 or dimerization domain (DD). Either peptide should disrupt the complex of a PSM dimer linked to IR via SH2 domains as proposed for PSM activation of tyrosine kinase JAK2. Either peptide abolished downstream insulin responses indistinguishable from PSM siRNA knockdown. Our results implicate an essential role of the PSM variants in the activation of the IR kinase and the resulting metabolic insulin response. PSM variants act as internal IR ligands that in addition to potentiating the insulin response stimulate IR catalytic activation even in the absence of insulin.     

Src homology domains of v-Src stabilize an active conformation of the tyrosine kinase catalytic domain

To examine the interactions between Src homology,domains and the tyrosine kinase catalytic domain of v-Src, various combinations of domains have been expressed in bacteria as fusion proteins. Constructs containing the isolated catalytic domain, SH2 + catalytic domain, and SH3 + SH2 + catalytic domains were active in autophosphorylation assays. For the catalytic domain of v-Src, but not for v-Abl, addition of exogenous Src SH3-SH2 domains stimulated the autophosphorylation activity. In contrast to results for autophosphorylation, constructs containing Src homology domains were more active towards a synthetic peptide substrate than the isolated catalytic domain. The ability of the SH2 and SH3 domains of v-Src to stabilize an active enzyme conformation was also confirmed by refolding after denaturation in guanidinium hydrochloride. Collectively the data suggest that, in addition to their roles in intermolecular protein-protein interactions, the Src homology regions of v-Src exert a positive influence on tyrosine kinase function, potentially by maintaining an active conformation of the catalytic domain.     

PSM/SH2-B distributes selected mitogenic receptor signals to distinct components in the PI3-kinase and MAP kinase signaling pathways

The Pro-rich, PH, and SH2 domain containing mitogenic signaling adapter PSM/SH2-B has been implicated as a cellular partner of various mitogenic receptor tyrosine kinases and related signaling mechanisms. Here, we report in a direct comparison of three peptide hormones, that PSM participates in the assembly of distinct mitogenic signaling complexes in response to insulin or IGF-I when compared to PDGF in cultured normal fibroblasts. The complex formed in response to insulin or IGF-I involves the respective peptide hormone receptor and presumably the established components leading to MAP kinase activation. However, our data suggest an alternative link from the PDGF receptor via PSM directly to MEK1/2 and consequently also to p44/42 activation, possibly through a scaffold protein. At least two PSM domains participate, the SH2 domain anticipated to link PSM to the respective receptor and the Pro-rich region in an association with an unidentified downstream component resulting in direct MEK1/2 and p44/42 regulation. The PDGF receptor signaling complex formed in response to PDGF involves PI 3-kinase in addition to the same components and interactions as described for insulin or IGF-I. PSM associates with PI 3-kinase via p85 and in addition the PSM PH domain participates in the regulation of PI 3-kinase activity, presumably through membrane interaction. In contrast, the PSM Pro-rich region appears to participate only in the MAP kinase signal. Both pathways contribute to the mitogenic response as shown by cell proliferation, survival, and focus formation. PSM regulates p38 MAP kinase activity in a pathway unrelated to the mitogenic response.     

Domain interactions in protein tyrosine kinase Csk.

Csk (C-terminal Src kinase) is a protein tyrosine kinase that phosphorylates Src family member C-terminal tails, resulting in downregulation of Src family members. It is composed of three principal domains: an SH3 (Src homology 3) domain, an SH2 (Src homology 2) domain, and a catalytic domain. The impact of the noncatalytic domains on kinase catalysis was investigated. The Csk catalytic domain was expressed in Escherichia coli as a recombinant glutathione S-transferase-fusion protein and demonstrated to have 100-fold reduced catalytic efficiency. Production of the catalytic domain by proteolysis of full-length Csk afforded a similar rate reduction. This suggested that the reduction in catalytic efficiency of the recombinant catalytic domain was intrinsic to the sequence and not an artifact related to faulty expression. This rate reduction was similar for peptide and protein substrates and was due almost entirely to a reduced k(cat) rather than to effects on substrate K(m)s. Viscosity experiments on the catalytic fragment kinase reaction demonstrated that the chemical (phosphoryl transfer) step had a reduced rate. While the Csk SH2 domain had no intermolecular effect on the kinase activity of the Csk catalytic domain, the SH3 domain and SH3-SH2 fragment led to a partial rescue (4-5-fold) of the lost kinase activity. This rescue was not achieved with two other SH3 domains (lymphoid cell kinase, Abelson kinase). The extrapolated K(d) of interaction for the Csk catalytic domain with the Csk SH3 domain was 2.2 microM and that of the Csk catalytic domain with the Csk SH3-SH2 fragment was 8.8 microM. Taken together, these findings suggest that there is likely an intramolecular interaction between the catalytic and SH3 domains in full-length Csk that is important for efficient catalysis. By employing a Csk SH3 specific type II polyproline helix peptide and carrying out site-directed mutagenesis, it was established that the SH3 surface that interacts with the catalytic domain was distinct from the surface that binds type II polyproline helix peptides. This finding suggests a novel mode of protein-protein interaction for an SH3 domain. The implications for Csk substrate selectivity, regulation, and function are discussed.     

Mutations in the N-terminal regulatory region reduce the catalytic activity of Csk, but do not affect its recognition of Src.

In addition to the C-terminal catalytic domain, Csk is a protein tyrosine kinase that has an N-terminal regulatory region that contains SH3 and SH2 domains. The role this region plays relative to the function of the catalytic domain is not clear. To study its role, we introduced either deletion or site-specific mutations within this region and analyzed the effect of such mutations on the catalytic activity of Csk and its ability to phosphorylate/inactivate Src protein tyrosine kinase, its physiological substrate in the cell. Deletion of the SH3 domain and the SH2 domain resulted in reductions of kinase activity by 70 and 96%, respectively. Mutations within the SH2 domain that abolished its ability to bind phosphotyrosine did not result in a significant loss of kinase activity. Mutation of Ser78 to Asp, located between the SH3 and the SH2 domains, resulted in a reduction of over 90% of the catalytic activity. The reduction in specific activity is not the result of any apparent physical instability of the mutants. Kinetic analyses indicate that the mutations did not affect the Km values for ATP-Mg or the polypeptide substrate. The ability of the mutants to phosphorylate and inactivate Src is directly correlated to their kinase activity. These results indicate that the regulatory region is important in optimizing the kinase activity of the catalytic domain, but apparently plays no direct or specific role in substrate recognition.     

Crystal Structure of the Src Family Kinase Hck SH3-SH2 Linker Regulatory Region Supports an SH3-dominant Activation Mechanism

Most mammalian cell types depend on multiple Src family kinases (SFKs) to regulate diverse signaling pathways. Strict control of SFK activity is essential for normal cellular function, and loss of kinase regulation contributes to several forms of cancer and other diseases. Previous x-ray crystal structures of the SFKs c-Src and Hck revealed that intramolecular association of their Src homology (SH) 3 domains and SH2 kinase linker regions has a key role in down-regulation of kinase activity. However, the amino acid sequence of the Hck linker represents a suboptimal ligand for the isolated SH3 domain, suggesting that it may form the polyproline type II helical conformation required for SH3 docking only in the context of the intact structure. To test this hypothesis directly, we determined the crystal structure of a truncated Hck protein consisting of the SH2 and SH3 domains plus the linker. Despite the absence of the kinase domain, the structures and relative orientations of the SH2 and SH3 domains in this shorter protein were very similar to those observed in near full-length, down-regulated Hck. However, the SH2 kinase linker adopted a modified topology and failed to engage the SH3 domain. This new structure supports the idea that these noncatalytic regions work together as a “conformational switch” that modulates kinase activity in a manner unique to the SH3 domain and linker topologies present in the intact Hck protein. Our results also provide fresh structural insight into the facile induction of Hck activity by HIV-1 Nef and other Hck SH3 domain binding proteins and implicate the existence of innate conformational states unique to individual Src family members that “fine-tune” their sensitivities to activation by SH3-based ligands.     

The evolutionarily conserved arrangement of domains in SRC family kinases is important for substrate recognition

The SH3-SH2-kinase domain arrangement in nonreceptor tyrosine kinases has been conserved throughout evolution. For Src family kinases, the relative positions of the domains are important for enzyme regulation; they permit the assembly of Src kinases into autoinhibited conformations. The SH3 and SH2 domains of Src family kinases have an additional role in determining the substrate specificity of the kinase. We addressed the question of whether the domain arrangement of Src family kinases has a role in substrate specificity by producing mutants with alternative arrangements. Our results suggest that changes in the positions of domains can lead to specific changes in the phosphorylation of Sam68 and Cas by Src. Phosphorylation of Cas by several mutants triggers downstream signaling leading to cell migration. The placement of the SH2 domain with respect to the catalytic domain of Src appears to be especially important for proper substrate recognition, while the placement of the SH3 domain is more flexible. The results suggest that the involvement of the SH3 and SH2 domains in substrate recognition is one reason for the strict conservation of the SH3-SH2-kinase architecture.     

Insulin receptor kinase‐independent signaling via tyrosine phosphorylation of phosphatase PHLPP1

Most insulin responses correlate well with insulin receptor (IR) Tyr kinase activation; however, critical exceptions to this concept have been presented. Specific IR mutants and stimulatory IR antibodies demonstrate a lack of correlation between IR kinase activity and specific insulin responses in numerous independent studies. IR conformation changes in response to insulin observed with various IR antibodies define an IR kinase‐independent signal that alters the C‐terminus. IR‐related receptors in lower eukaryotes that lack a Tyr kinase point to an alternative mechanism of IR signaling earlier in evolution. However, the implied IR kinase‐independent signaling mechanism remained obscure at the molecular level. Here we begin to define the molecular basis of an IR‐dependent but IR kinase‐independent insulin signal that is equally transmitted by a kinase‐inactive mutant IR. This insulin signal results in Tyr phosphorylation and catalytic activation of phosphatase PHLPP1 via a PI 3‐kinase‐independent, wortmannin‐insensitive signaling pathway. Dimerized SH2B1/PSM is a critical activator of the IR kinase and the resulting established insulin signal. In contrast it is an inhibitor of the IR kinase‐independent insulin signal and disruption of SH2B1/PSM dimer binding to IR potentiates this signal. Dephosphorylation of Akt2 by PHLPP1 provides an alternative, SH2B1/PSM‐regulated insulin‐signaling pathway from IR to Akt2 of opposite polarity and distinct from the established PI 3‐kinase‐dependent signaling pathway via IRS proteins. In combination, both pathways should allow the opposing regulation of Akt2 activity at two phosphorylation sites to specifically define the insulin signal in the background of interfering Akt‐regulating signals, such as those controlling cell proliferation and survival. J. Cell. Biochem. 107: 65–75, 2009. © 2009 Wiley‐Liss, Inc.     

Effects of SH2 and SH3 deletions on the functional activities of wild-type and transforming variants of c-Src.

The amino-termina, noncatalytic half of Src contains two domains, designated the Src homology 2 (SH2) and Src homology 3 (SH3) domains, that are highly conserved among members of the Src family of tyrosine kinases. The SH2 domain (which can be further divided into the B and C homology boxes) and the SH3 domain (also referred to as the A box) are also found in several proteins otherwise unrelated to protein tyrosine kinases. It is believed that these domains are important for directing specific protein-protein interactions necessary for the proper functioning of Src. To determine the importance of the SH2 and SH3 domains in regulating the functions of c-Src, we evaluated mutants of c-Src lacking the A box (residues 88 to 137), the B box (residues 148 to 187) or the C box (residues 220 to 231). Each of these deletions caused a 14- to 30-fold increase in the in vitro level of kinase activity of c-Src. Chicken embryo fibroblasts expressing the deletion mutants displayed a transformed cell morphology, formed colonies in soft agar, and contained elevated levels of cellular phosphotyrosine-containing proteins. Src substrates p36, p85, p120, p125, the GTPase-activating protein (GAP), and several GAP-associated proteins were phosphorylated on tyrosine in cells expressing the A, B, or C box deletion mutant. p110 was highly phosphorylated in cells expressing the C box mutant, was weakly phosphorylated in cells expressing the B box mutant, and was not phosphorylated in cells expressing the A box mutant. Expression of the mutant proteins caused a reorganization of the actin cytoskeleton similar to that seen in v-Src-transformed cells. In addition, deletion of the A, B, or C box did not diminish the transforming or enzymatic activity of an activated variant of c-Src, E378G. These data indicate that deletion of the A, B, or C homology box causes an activation of the catalytic and transforming potential of c-Src and that while these mutations caused subtle differences in substrate phosphorylation, the homology boxes are not required for many of the phenotypic changes associated with transformation by Src.     

Specific interactions of neuronal focal adhesion kinase isoforms with Src kinases and amphiphysin

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that activates Src family kinases via SH2- and SH3-mediated interactions. Specific FAK isoforms (FAK+), responsive to depolarization and neurotransmitters, are enriched in neurons. We analyzed the interactions of endogenous FAK+ and recombinant FAK+ isoforms containing amino acid insertions (boxes 6,7,28) with an array of SH3 domains and the c-Src SH2/SH3 domain tandem. Endogenous FAK+ bound specifically to the SH3 domains of c-Src (but not n-Src), Fyn, Yes, phosphtidylinositol-3 kinase, amphiphysin II, amphiphysin I, phospholipase Cgamma and NH2-terminal Grb2. The inclusion of boxes 6,7 was associated with a significant decrease in the binding of FAK+ to the c-Src and Fyn SH3 domains, and a significant increase in the binding to the Src SH2 domain, as a consequence of the higher phosphorylation of Tyr-397. The novel interaction with the amphiphysin SH3 domain, involving the COOH-terminal proline-rich region of FAK, was confirmed by coimmunoprecipitation of the two proteins and a closely similar response to stimuli affecting the actin cytoskeleton. Moreover, an impairment of endocytosis was observed in synaptosomes after internalization of a proline-rich peptide corresponding to the site of interaction. The data account for the different subcellular distribution of FAK and Src kinases and the specific regulation of the transduction pathways linked to FAK activation in the brain and implicate FAK in the regulation of membrane trafficking in nerve terminals.     

A peptide model system for processive phosphorylation by Src family kinases

The Src homology 2 (SH2) and Src homology 3 (SH3) domains of Src family kinases are involved in substrate recognition in vivo. Many cellular substrates of Src kinases contain a large number of potential phosphorylation sites, and the SH2 and SH3 domains of Src are known to be required for phosphorylation of these substrates. In principle, Src could phosphorylate these substrates by either a processive mechanism, in which the enzyme remains bound to the peptide substrate during multiple phosphorylation events, or a nonprocessive (distributive) mechanism, where each phosphorylation requires a separate binding interaction between enzyme and substrate. Here we use a synthetic peptide system to demonstrate that Hck, a Src family kinase, can phosphorylate substrates containing an SH2 domain ligand by a processive mechanism. Hck catalyzes the phosphorylation of these sites in a defined order. Furthermore, we show that addition of an SH3 domain to a peptide can enhance its phosphorylation both by activating Hck and by increasing the affinity of the substrate. On the basis of our observations on the role of the SH2 and SH3 domains in substrate recognition, we present a model for substrate targeting in vivo.     

Structural basis for domain-domain communication in a protein tyrosine kinase, the C-terminal Src kinase

The catalytic activity of protein tyrosine kinases is commonly regulated by domain-domain interactions. The C-terminal Src kinase (Csk) contains a catalytic domain and the regulatory SH3 and SH2 domains. Both the presence of the regulatory domains and binding of specific phosphotyrosine-containing proteins to the SH2 domain activate Csk. The structural basis for both modes of activation is investigated here. First, the SH3-SH2 linker is crucial for Csk activation. Mutagenic and kinetic studies demonstrate that this activation is mediated by a cation-pi interaction between Arg68 and Trp188. Second, Ala scanning and kinetic analyses on residues in the SH2-catalytic domain interface identify three functionally distinct types of residues in mediating the communication between the SH2 and the catalytic domains. Type I residues are important in mediating a ligand-triggered activation of Csk because their mutation severely reduces Csk activation by the SH2 domain ligand. Type II residues are involved in suppressing Csk activity, and their mutation activates Csk, but makes Csk less sensitive to activation by the SH2 ligand. Both type I and type II residues are likely involved in mediating SH2 ligand-triggered activation of Csk. Type III residues are those located in the SH2 domain whose mutation severely decreases Csk catalytic activity without affecting the SH2 ligand-triggered activation. These residues likely mediate SH2 activation of Csk regardless of SH2-ligand interaction. These studies lead us to propose a domain-domain communication model that provides functional insights into the topology of Csk family of protein tyrosine kinases.     

Mitogenic roles of Gab1 and Grb10 as direct cellular partners in the regulation of MAP kinase signaling

Functions of signaling mediators Grb10 or Gab1 have been described in mitogenesis but remained disconnected. Here, we report the peptide hormone-dependent direct association between Grb10 and Gab1 and their functional connection in mitogenic signaling via MAP kinase using cultured fibroblasts as a model. In response to PDGF-, IGF-I, or insulin increased levels of Grb10 potentiated cell proliferation or survival whereas dominant-negative, domain-specific Grb10 peptide mimetics attenuated cell proliferation. This response was sensitive to p44/42 MAPK inhibitor but not to p38 MAPK inhibitor. In response to IGF-I or insulin Raf-1, MEK 1/2, and p44/42 MAPK were regulated by Grb10 but not Ras or p38 MAPK. In response to PDGF MEK 1/2, p44/42 MAPK and p38 MAPK were regulated by Grb10 but not Ras or Raf-1. Peptide hormone-dependent co-immunoprecipitation of Grb10 and Gab1 was demonstrated and specifically blocked by a Grb10 SH2 domain peptide mimetic. This domain was sufficient for direct, peptide hormone-dependent association with Gab1 via the Crk binding region. In response to PDGF, IGF-I, or insulin, in a direct comparison, elevated levels of mouse Grb10 delta, or human Grb10 beta or zeta equally potentiated fibroblast proliferation. Proliferation was severely reduced by Gab1 gene disruption whereas an elevated Gab1 gene dose proportionally stimulated Grb10-potentiated cell proliferation. In conclusion, Gab1 and Grb10 function as direct binding partners in the regulation of the mitogenic MAP kinase signal. In cultured fibroblasts, elevated levels of human Grb10 beta, zeta or mouse Grb10 delta comparably potentiate mitogenesis in response to PDGF, IGF-I, or insulin.     

The Abl SH2-kinase linker naturally adopts a conformation competent for SH3 domain binding

The core of the Abelson tyrosine kinase (c-Abl) is structurally similar to Src-family kinases where SH3 and SH2 domains pack against the backside of the kinase domain in the down-regulated conformation. Both kinase families depend upon intramolecular association of SH3 with the linker joining the SH2 and kinase domains for suppression of kinase activity. Hydrogen deuterium exchange (HX) and mass spectrometry (MS) were used to probe intramolecular interaction of the c-Abl SH3 domain with the linker in recombinant constructs lacking the kinase domain. Under physiological conditions, the c-Abl SH3 domain undergoes partial unfolding, which is stabilized by ligand binding, providing a unique assay for SH3:linker interaction in solution. Using this approach, we observed dynamic association of the SH3 domain with the linker in the absence of the kinase domain. Truncation of the linker before W254 completely prevented cis-interaction with SH3, while constructs containing amino acids past this point showed SH3:linker interactions. The observation that the Abl linker sequence exhibits SH3-binding activity in the absence of the kinase domain is unique to Abl and was not observed with Src-family kinases. These results suggest that SH3:linker interactions may have a more prominent role in Abl regulation than in Src kinases, where the down-regulated conformation is further stabilized by a second intramolecular interaction between the C-terminal tail and the SH2 domain.     

Biochemical properties of the Cdc42-associated tyrosine kinase ACK1. Substrate specificity, authphosphorylation, and interaction with Hck

ACK1 (activated Cdc42-associated kinase 1) is a nonreceptor tyrosine kinase and the only tyrosine kinase known to interact with Cdc42. To characterize the enzymatic properties of ACK, we have expressed and purified active ACK using the baculovirus/Sf9 cell system. This ACK1 construct contains (from N to C terminus) the kinase catalytic domain, SH3 domain, and Cdc42-binding Cdc42/Rac interactive binding (CRIB) domain. We characterized the substrate specificity of ACK1 using synthetic peptides, and we show that the specificity of the ACK1 catalytic domain most closely resembles that of Abl. Purified ACK1 undergoes autophosphorylation, and autophosphorylation enhances kinase activity. We identified Tyr284 in the activation loop of ACK1 as the primary autophosphorylation site using mass spectrometry. When expressed in COS-7 cells, the Y284F mutant ACK1 showed dramatically reduced levels of tyrosine phosphorylation. Although the SH3 and CRIB domains of purified ACK1 are able to bind ligands (a polyproline peptide and Cdc42, respectively), the addition of ligands did not stimulate tyrosine kinase activity. To characterize potential interacting partners for ACK1, we screened several SH2 and SH3 domains for their ability to bind to full-length ACK1 or to the catalytic-SH3-CRIB construct. ACK1 interacts most strongly with the SH3 domains of Src family kinases (Src or Hck) via its C-terminal proline-rich domain. Co-expression of Hck with kinase-inactive ACK1(K158R) in mammalian cells resulted in tyrosine phosphorylation of ACK1, suggesting that ACK1 is a substrate for Hck. Our data suggest that Hck is a novel binding partner for ACK1 that can regulate ACK1 activity by phosphorylation.     

Artemin activates axonal growth via SFK and ERK-dependent signalling pathways in mature dorsal root ganglia neurons

Artemin, one of the glial cell line-derived neurotrophic factor (GDNF) family, enhances the generation and survival of early sympathetic neurons and superior cervical ganglion (SCG) neurons. Src-family kinases (SFK) are involved in the growth and differentiation of cells, which are composed of unique Src homology 2 (SH2), Src homology 3 (SH3) and kinase domains. Various extra-cellular molecules containing growth factors and G-protein coupled receptors stimulate SFK. In this report, artemin is shown to have a significant effect on the neurite growth of dorsal root ganglia (DRG) neurons. Also, artemin triggers Src-family kinase activation and the phosphorylation of extra-cellular signal-regulated kinases (ERK) mitogen-activated protein kinase (MAPK). Artemin also regulated actin polymerization. There are several indications that another SH3-containing protein, Hck, and an SH3-containing adaptor protein, Nck1, play an important role in the organization of the actin cytoskeleton by cellular signalling. These findings suggest that the exploration of binding partners for the SH3 domain could provide an insight into regulation between the microtubule and actin networks. The binding partners for the SH3 domains of Nck, Src and Hck that we identified were Smc chromosome segregation ATPases, FOG Zn-finger protein and the FYVE zinc-binding domain, respectively.     

Genome wide analysis of pathogenic SH2 domain mutations

The authors have made a genome-wide analysis of mutations in Src homology 2 (SH2) domains associated with human disease. Disease-causing mutations have been detected in the SH2 domains of cytoplasmic signaling proteins Bruton tyrosine kinase (BTK), SH2D1A, Ras GTPase activating protein (RasGAP), ZAP-70, SHP-2, STAT1, STAT5B, and the p85alpha subunit of the PIP3. Mutations in the BTK, SH2D1A, ZAP70, STAT1, and STAT5B genes have been shown to cause diverse immunodeficiencies, whereas the mutations in RASA1 and PIK3R1 genes lead to basal carcinoma and diabetes, respectively. PTPN11 mutations cause Noonan sydrome and different types of cancer, depending mainly on whether the mutation is inherited or sporadic. We collected and analyzed all known pathogenic mutations affecting human SH2 domains by bioinformatics methods. Among the investigated protein properties are sequence conservation and covariance, structural stability, side chain rotamers, packing effects, surface electrostatics, hydrogen bond formation, accessible surface area, salt bridges, and residue contacts. The majority of the mutations affect positions essential for phosphotyrosine ligand binding and specificity. The structural basis of the SH2 domain diseases was elucidated based on the bioinformatic analysis.