Identification of a New Interaction Mode between the Src Homology 2 Domain of C-terminal Src Kinase (Csk) and Csk-binding Protein/Phosphoprotein Associated with Glycosphingolipid Microdomains

Proteins with Src homology 2 (SH2) domains play major roles in tyrosine kinase signaling. Structures of many SH2 domains have been studied, and the regions involved in their interactions with ligands have been elucidated. However, these analyses have been performed using short peptides consisting of phosphotyrosine followed by a few amino acids, which are described as the canonical recognition sites. Here, we report the solution structure of the SH2 domain of C-terminal Src kinase (Csk) in complex with a longer phosphopeptide from the Csk-binding protein (Cbp). This structure, together with biochemical experiments, revealed the existence of a novel binding region in addition to the canonical phosphotyrosine 314-binding site of Cbp. Mutational analysis of this second region in cells showed that both canonical and novel binding sites are required for tumor suppression through the Cbp-Csk interaction. Furthermore, the data indicate an allosteric connection between Cbp binding and Csk activation that arises from residues in the βB/βC loop of the SH2 domain.     

Roles of the SH2 and SH3 domains in the regulation of neuronal Src kinase functions

Previous studies demonstrated that intra-domain interactions between Src family kinases (SFKs), stabilized by binding of the phosphorylated C-terminus to the SH2 domain and/or binding of the SH2 kinase linker to the SH3 domain, lock the molecules in a closed conformation, disrupt the kinase active site, and inactivate SFKs. Here we report that the up-regulation of N-methyl-D-aspartate receptors (NMDARs) induced by expression of constitutively active neuronal Src (n-Src), in which the C-terminus tyrosine is mutated to phenylalanine (n-Src/Y535F), is significantly reduced by dysfunctions of the SH2 and/or SH3 domains of the protein. Furthermore, we found that dysfunctions of SH2 and/or SH3 domains reduce auto-phosphorylation of the kinase activation loop, depress kinase activity, and decrease NMDAR phosphorylation. The SH2 domain plays a greater regulatory role than the SH3 domain. Our data also show that n-Src binds directly to the C-terminus of the NMDAR NR2A subunit in vitro, with a K(D) of 108.2 ± 13.3 nM. This binding is not Src kinase activity-dependent, and dysfunctions of the SH2 and/or SH3 domains do not significantly affect the binding. These data indicate that the SH2 and SH3 domains may function to promote the catalytic activity of active n-Src, which is important in the regulation of NMDAR functions.     

SH3-dependent stimulation of Src-family kinase autophosphorylation without tail release from the SH2 domain in vivo

Src family protein-tyrosine kinase activity is suppressed by two intramolecular interactions. These involve binding of the SH2 domain to the phosphorylated C-terminal tail and association of the SH3 domain with a polyproline type II helix formed by the SH2-kinase linker. Here we show that SH3-dependent activation of the Src family member Hck by HIV-1 Nef binding or by SH2-kinase linker mutation does not affect tail tyrosine phosphorylation in fibroblasts. Surprisingly, replacement of the wild type Hck tail with a high-affinity SH2 domain-binding sequence did not affect Hck activation or downstream signaling by these SH3-dependent mechanisms, suggesting that activation through SH3 occurs without SH2-tail dissociation. These results identify SH3-linker interaction as an independent mode of Hck kinase regulation in vivo and suggest that different mechanisms of Src kinase activation may generate distinct output signals because of differences in SH2 or SH3 domain accessibility.     

C-terminal Src kinase-homologous kinase (CHK), a unique inhibitor inactivating multiple active conformations of Src family tyrosine kinases

The Src family of protein kinases (SFKs) mediates mitogenic signal transduction, and constitutive SFK activation is associated with tumorigenesis. To prevent constitutive SFK activation, the catalytic activity of SFKs in normal mammalian cells is suppressed mainly by two inhibitors called C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK), which inactivate SFKs by phosphorylating a consensus tyrosine near the C terminus of SFKs (Y(T)). The phosphorylated Y(T) intramolecularly binds to the SH2 domain of SFKs. This interaction, known as pY(T)/SH2 interaction, together with binding between the SH2 kinase linker and the SH3 domain of SFKs (linker/SH3 interaction) stabilizes SFKs in a "closed" inactive conformation. We previously discovered an alternative mechanism CHK employs to inhibit SFKs. This mechanism, referred to as the non-catalytic inhibitory mechanism, involves tight binding of CHK to SFKs; the binding alone is sufficient to inhibit SFKs. Herein, we constructed multiple active conformations of an SFK member, Hck, by systematically disrupting the two inhibitory interactions. We found that CHK employs the non-catalytic mechanism to inactivate these active conformations of Hck. However, CHK does not bind Hck when it adopts the inactive conformation in which both inhibitory interactions are intact. These data indicate that binding of CHK to SFKs via the non-catalytic mechanism is governed by the conformations of SFKs. Although CSK is also an inhibitor of SFKs, it does not inhibit SFKs by a similar non-catalytic mechanism. Thus, the non-catalytic inhibitory mechanism is a unique property of CHK that allows it to down-regulate multiple active conformations of SFKs.     

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.     

Differential Sensitivity of Src-Family Kinases to Activation by SH3 Domain Displacement

Src-family kinases (SFKs) are non-receptor protein-tyrosine kinases involved in a variety of signaling pathways in virtually every cell type. The SFKs share a common negative regulatory mechanism that involves intramolecular interactions of the SH3 domain with the PPII helix formed by the SH2-kinase linker as well as the SH2 domain with a conserved phosphotyrosine residue in the C-terminal tail. Growing evidence suggests that individual SFKs may exhibit distinct activation mechanisms dictated by the relative strengths of these intramolecular interactions. To elucidate the role of the SH3:linker interaction in the regulation of individual SFKs, we used a synthetic SH3 domain-binding peptide (VSL12) to probe the sensitivity of downregulated c-Src, Hck, Lyn and Fyn to SH3-based activation in a kinetic kinase assay. All four SFKs responded to VSL12 binding with enhanced kinase activity, demonstrating a conserved role for SH3:linker interaction in the control of catalytic function. However, the sensitivity and extent of SH3-based activation varied over a wide range. In addition, autophosphorylation of the activation loops of c-Src and Hck did not override regulatory control by SH3:linker displacement, demonstrating that these modes of activation are independent. Our results show that despite the similarity of their downregulated conformations, individual Src-family members show diverse responses to activation by domain displacement which may reflect their adaptation to specific signaling environments in vivo.     

Enhanced SH3/Linker Interaction Overcomes Abl Kinase Activation by Gatekeeper and Myristic Acid Binding Pocket Mutations and Increases Sensitivity to Small Molecule Inhibitors

Multidomain kinases such as c-Src and c-Abl are regulated by complex allosteric interactions involving their noncatalytic SH3 and SH2 domains. Here we show that enhancing natural allosteric control of kinase activity by SH3/linker engagement has long-range suppressive effects on the kinase activity of the c-Abl core. Surprisingly, enhanced SH3/linker interaction also dramatically sensitized the Bcr-Abl tyrosine kinase associated with chronic myelogenous leukemia to small molecule inhibitors that target either the active site or the myristic acid binding pocket in the kinase domain C-lobe. Dynamics analyses using hydrogen exchange mass spectrometry revealed a remarkable allosteric network linking the SH3 domain, the myristic acid binding pocket, and the active site of the c-Abl core, providing a structural basis for the biological observations. These results suggest a rational strategy for enhanced drug targeting of Bcr-Abl and other multidomain kinase systems that use multiple small molecules to exploit natural mechanisms of kinase control.     

Affinity of Src family kinase SH3 domains for HIV Nef in vitro does not predict kinase activation by Nef in vivo

Nef is an HIV accessory protein required for high-titer viral replication and AIDS progression. Previous studies have shown that the SH3 domains of Hck and Lyn bind to Nef via proline-rich sequences in vitro, identifying these Src-related kinases as potential targets for Nef in vivo. Association of Nef with Hck causes displacement of the intramolecular interaction between the SH3 domain and the SH2-kinase linker, leading to kinase activation both in vitro and in vivo. In this study, we investigated whether interaction with Nef induces activation of other Src family kinases (Lyn, Fyn, Src, and Lck) following coexpression with Nef in Rat-2 fibroblasts. Coexpression with Nef induced Hck kinase activation and fibroblast transformation, consistent with previous results. In contrast, coexpression of Nef with Lyn was without effect, despite equivalent binding of Nef to full-length Lyn and Hck. Furthermore, Nef was found to suppress the kinase and transforming activities of Fyn, the SH3 domain of which exhibits low affinity for Nef. Coexpression with Nef did not alter c-Src or Lck tyrosine kinase or transforming activity in this system. Differential modulation of Src family members by Nef may produce unique downstream signals depending on the profile of Src kinases expressed in a given cell type.     

Activation of the Src family kinase Hck without SH3-linker release

Src family protein-tyrosine kinases are regulated by intramolecular binding of the SH2 domain to the C-terminal tail and association of the SH3 domain with the SH2 kinase-linker. The presence of two regulatory interactions raises the question of whether disruption of both is required for kinase activation. To address this question, we engineered a high affinity linker (HAL) mutant of the Src family member Hck in which an optimal SH3 ligand was substituted for the natural linker. Surface plasmon resonance analysis demonstrated tight intramolecular binding of the modified HAL sequence to SH3. Hck-HAL was then combined with a tail tyrosine mutation (Y501F) and expressed in Rat-2 fibroblasts. Surprisingly, Hck-HAL-Y501F showed strong transforming and kinase activities, demonstrating that intramolecular SH3-linker release is not required for SH2-based kinase activation. In Saccharomyces cerevisiae, which lacks the negative regulatory tail kinase Csk, wild-type Hck was more strongly activated in the presence of an SH3-binding protein (human immunodeficiency virus-1 Nef), indicating persistence of native SH3-linker interaction in an active Hck conformation. Taken together, these data support the existence of multiple active conformations of Src family kinases that may generate unique downstream signals.     

Intramolecular binding of a proximal PPII helix to an SH3 domain in the fusion protein SH3Hck: PPIIhGAP

SH3 domains are a conserved feature of many nonreceptor protein tyrosine kinases, such as Hck, and often function in substrate recruitment and regulation of kinase activity. SH3 domains modulate kinase activity by binding to polyproline helices (PP_II helix) either intramolecularly or in target proteins. The preponderance of bimolecular and distal interactions between SH3 domains and PP_II helices led us to investigate whether proximal placement of a PP_II helix relative to an SH3 domain would result in tight, intramolecular binding. We have fused the PP_II helix region of human GAP to the C-terminus of Hck SH3 and expressed the recombinant fusion protein in Eschericheria coli. The fusion protein, SH3_Hck: PPII_hGAP, folded spontaneously into a structure in which the PP_II helix was bound intramolecularly to the hydrophobic crevice of the SH3 domain. The SH3_Hck: PPII_hGAP fusion protein is useful for investigating SH3: PP_II helix interactions, for studying concepts in protein folding and design, and may represent a protein structural motif that is widely distributed in nature.     

The SH3 domain of Src can downregulate its kinase activity in the absence of the SH2 domain-pY527 interaction

The contact between the SH2 domain and the C-terminal tail of c-Src inhibits its kinase activity via a complex network of interactions, including the SH3 domain. We examined the role of the SH3 domain in v-Src, where the C-terminal tail is mutated and unbound. We used the v-Src variants Prague C (PRC) and Schmidt-Ruppin A (SRA), which are of low and high kinase activities, respectively, to measure phosphorylation in vitro by immunoprecipitated kinases produced in Saccharomyces cerevisiae. Swapping the regulatory domains between SRA and PRC revealed that N117D, I96T, and V124L mutations in the n-src- and RT-loops of the SH3 domain of PRC are responsible for the low kinase activity of PRC. Moreover, introducing D117N, R95W, T96I, and L124V into activated c-Src(Y527F) caused a 2.5-fold increase in its activity. The mutations in the CD linker KP249,250DG and L255A, which were shown to activate c-Src, had no effect on the activity of the "SH2-activated" Src kinases. Together our data suggest that in the "SH2-activated" forms of Src, the SH3 domain continues to influence the kinase activity via the direct contacts of the n-src- and RT-loops with the kinase N-terminal lobe.     

Proline Isomerization Preorganizes the Itk SH2 Domain for Binding to the Itk SH3 Domain

We report here the NMR-derived structure of the binary complex formed by the interleukin-2 tyrosine kinase (Itk) Src homology 3 (SH3) and Src homology 2 (SH2) domains. The interaction is independent of both a phosphotyrosine motif and a proline-rich sequence, the classical targets of the SH2 and SH3 domains, respectively. The Itk SH3/SH2 structure reveals the molecular details of this nonclassical interaction and provides a clear picture for how the previously described prolyl cis/trans isomerization present in the Itk SH2 domain mediates SH3 binding. The higher-affinity cis SH2 conformer is preorganized to form a hydrophobic interface with the SH3 domain. The structure also provides insight into how autophosphorylation in the Itk SH3 domain might increase the affinity of the intermolecular SH3/SH2 interaction. Finally, we can compare this Itk complex with other examples of SH3 and SH2 domains engaging their ligands in a nonclassical manner. These small binding domains exhibit a surprising level of diversity in their binding repertoires.     

A novel disulfide bond in the SH2 Domain of the C-terminal Src kinase controls catalytic activity

The SH2 domain of the C-terminal Src kinase [Csk] contains a unique disulfide bond that is not present in other known SH2 domains. To investigate whether this unusual disulfide bond serves a novel function, the effects of disulfide bond formation on catalytic activity of the full-length protein and on the structure of the SH2 domain were investigated. The kinase activity of full-length Csk decreases by an order of magnitude upon formation of the disulfide bond in the distal SH2 domain. NMR spectra of the fully oxidized and fully reduced SH2 domains exhibit similar chemical shift patterns and are indicative of similar, well-defined tertiary structures. The solvent-accessible disulfide bond in the isolated SH2 domain is highly stable and far from the small lobe of the kinase domain. However, reduction of this bond results in chemical shift changes of resonances that map to a cluster of residues that extend from the disulfide bond across the molecule to a surface that is in direct contact with the small lobe of the kinase domain in the intact molecule. Normal mode analyses and molecular dynamics calculations suggest that disulfide bond formation has large effects on residues within the kinase domain, most notably within the active-site cleft. Overall, the data indicate that reversible cross-linking of two cysteine residues in the SH2 domain greatly impacts catalytic function and interdomain communication in Csk.     

Bacterial expression and purification of active hematopoietic cell kinase

Src family kinases (SFKs) are traditionally purified from eukaryotic expression systems. These expression systems can be costly, yield heterogeneously phosphorylated protein samples and present difficulties when metabolic labeling is required for structural studies. Therefore, many attempts have been made to develop bacterial purification systems for SFKs. So far, high-yield bacterial expression systems have only been achieved for SFK kinase domains or for inactive mutants of constructs containing the regulatory SH3 and SH2 domains, but not for their active forms. Herein described is a bacterial expression system for the wild type, active SFK Hck containing SH3, SH2 and kinase domains. Hck plays an important role in phagocyte function as well as the etiology of chronic myeloid leukemia as Hck is an interaction partner of Bcr-Abl. Structural studies of Hck are essential to fully understand the signaling processes involved in host defense and leukemogenesis. Successful bacterial expression of Hck was possible by a dual strategy: (1) co-expression with YopH phosphatase in order to control host toxicity, and (2) expression in a bacterial strain that is RNase E deficient, which dramatically increased overall expression levels. The expressed Hck construct is unphosphorylated and appears to be in an open conformation. Bacterially expressed Hck is capable of autophosphorylation, phosphorylates substrate at rates comparable to insect cell expressed Hck, and can be inhibited by staurosporine and Csk.     

Two Amino Acid Residues Confer Different Binding Affinities of Abelson Family Kinase Src Homology 2 Domains for Phosphorylated Cortactin

The closely related Abl family kinases, Arg and Abl, play important non-redundant roles in the regulation of cell morphogenesis and motility. Despite similar N-terminal sequences, Arg and Abl interact with different substrates and binding partners with varying affinities. This selectivity may be due to slight differences in amino acid sequence leading to differential interactions with target proteins. We report that the Arg Src homology (SH) 2 domain binds two specific phosphotyrosines on cortactin, a known Abl/Arg substrate, with over 10-fold higher affinity than the Abl SH2 domain. We show that this significant affinity difference is due to the substitution of arginine 161 and serine 187 in Abl to leucine 207 and threonine 233 in Arg, respectively. We constructed Abl SH2 domains with R161L and S187T mutations alone and in combination and find that these substitutions are sufficient to convert the low affinity Abl SH2 domain to a higher affinity “Arg-like” SH2 domain in binding to a phospho-cortactin peptide. We crystallized the Arg SH2 domain for structural comparison to existing crystal structures of the Abl SH2 domain. We show that these two residues are important determinants of Arg and Abl SH2 domain binding specificity. Finally, we expressed Arg containing an “Abl-like” low affinity mutant Arg SH2 domain (L207R/T233S) and find that this mutant, although properly localized to the cell periphery, does not support wild type levels of cell edge protrusion. Together, these observations indicate that these two amino acid positions confer different binding affinities and cellular functions on the distinct Abl family kinases.     

Mechanism of Phosphorylation-induced Activation of Phospholipase C-γ Isozymes

The lipase activity of most phospholipases C (PLCs) is basally repressed by a highly degenerate and mostly disordered X/Y linker inserted within the catalytic domain. Release of this auto-inhibition is driven by electrostatic repulsion between the plasma membrane and the electronegative X/Y linker. In contrast, PLC-γ isozymes (PLC-γ1 and -γ2) are structurally distinct from other PLCs because multiple domains are present in their X/Y linker. Moreover, although many tyrosine kinases directly phosphorylate PLC-γ isozymes to enhance their lipase activity, the underlying molecular mechanism of this activation remains unclear. Here we define the mechanism for the unique regulation of PLC-γ isozymes by their X/Y linker. Specifically, we identify the C-terminal SH2 domain within the X/Y linker as the critical determinant for auto-inhibition. Tyrosine phosphorylation of the X/Y linker mediates high affinity intramolecular interaction with the C-terminal SH2 domain that is coupled to a large conformational rearrangement and release of auto-inhibition. Consequently, PLC-γ isozymes link phosphorylation to phospholipase activation by elaborating upon primordial regulatory mechanisms found in other PLCs.     

Effect of the SH3-SH2 domain linker sequence on the structure of Hck kinase

The coordination of activity in biological systems requires the existence of different signal transduction pathways that interact with one another and must be precisely regulated. The Src-family tyrosine kinases, which are found in many signaling pathways, differ in their physiological function despite their high overall structural similarity. In this context, the differences in the SH3-SH2 domain linkers might play a role for differential regulation, but the structural consequences of linker sequence remain poorly understood. We have therefore performed comparative molecular dynamics simulations of wildtype Hck and of a mutant Hck in which the SH3-SH2 domain linker is replaced by the corresponding sequence from the homologous kinase Lck. These simulations reveal that linker replacement not only affects the orientation of the SH3 domain itself, but also leads to an alternative conformation of the activation segment in the Hck kinase domain. The sequence of the SH3-SH2 domain linker thus exerts a remote effect on the active site geometry and might therefore play a role in modulating the structure of the inactive kinase or in fine-tuning the activation process itself.     

The role of the linker between the SH2 domain and catalytic domain in the regulation and function of Src.

The crystal structures of the regulated Src and Hck tyrosine kinases show intramolecular interactions between the phosphorylated tail and the SH2 domain as well as between the SH3 domain, the SH2-catalytic domain linker (SH2-CD linker) and the catalytic domain. The relative contribution of these interactions to regulation of activity is poorly understood. Mutational analysis of Src and Lck revealed that interaction of the SH2-CD linker with the SH3 domain is crucial for regulation. Moreover, three sites of interaction of the linker with the catalytic domain, one at the beginning (Trp260) and two at the back of the small lobe, opposite the catalytic cleft (beta2/beta3 loop; alphaC/beta4 loop), impinge on Src activity. Other activating mutations map to the front of the catalytic domain in the loop preceding the alphaC-helix (beta3/alphaC loop). SH2-CD linker mutants are deregulated in mammalian cells but transform fibroblasts weakly, suggesting that the linker may bind cellular components. Interpretation of our results on the basis of the crystal structure of Src favours a model in which the correctly positioned SH2-CD linker exerts an inhibitory function on catalysis of Src family members by facilitating displacement of the alphaC-helix. This study may provide a template for the generation of deregulated versions of other protein kinases.