Adaptor Protein GRB2 Promotes Src Tyrosine Kinase Activation and Podosomal Organization by Protein-tyrosine Phosphatase ? in Osteoclasts

The non-receptor isoform of protein-tyrosine phosphatase ϵ (cyt-PTPe) supports adhesion of bone-resorbing osteoclasts by activating Src downstream of integrins. Loss of cyt-PTPe reduces Src activity in osteoclasts, reduces resorption of mineralized matrix both in vivo and in cell culture, and induces mild osteopetrosis in young female PTPe KO mice. Activation of Src by cyt-PTPe is dependent upon this phosphatase undergoing phosphorylation at its C-terminal Tyr-638 by partially active Src. To understand how cyt-PTPe activates Src, we screened 73 Src homology 2 (SH2) domains for binding to Tyr(P)-638 of cyt-PTPe. The SH2 domain of GRB2 bound Tyr(P)-638 of cyt-PTPe most prominently, whereas the Src SH2 domain did not bind at all, suggesting that GRB2 may link PTPe with downstream molecules. Further studies indicated that GRB2 is required for activation of Src by cyt-PTPe in osteoclast-like cells (OCLs) in culture. Overexpression of GRB2 in OCLs increased activating phosphorylation of Src at Tyr-416 and of cyt-PTPe at Tyr-638; opposite results were obtained when GRB2 expression was reduced by shRNA or by gene inactivation. Phosphorylation of cyt-PTPe at Tyr-683 and its association with GRB2 are integrin-driven processes in OCLs, and cyt-PTPe undergoes autodephosphorylation at Tyr-683, thus limiting Src activation by integrins. Reduced GRB2 expression also reduced the ability of bone marrow precursors to differentiate into OCLs and reduced the fraction of OCLs in which podosomal adhesion structures assume organization typical of active, resorbing cells. We conclude that GRB2 physically links cyt-PTPe with Src and enables cyt-PTPe to activate Src downstream of activated integrins in OCLs.     

Rac1 Recruits the Adapter Protein CMS/CD2AP to Cell-Cell Contacts

Rac1 is a member of the Rho family of small GTPases, which regulate cell adhesion and migration through their control of the actin cytoskeleton. Rho-GTPases are structurally very similar, with the exception of a hypervariable domain in the C terminus. Using peptide-based pulldown assays in combination with mass spectrometry, we previously showed that the hypervariable domain in Rac1 mediates specific protein-protein interactions. Most recently, we found that the Rac1 C terminus associates to the ubiquitously expressed adapter protein CMS/CD2AP. CD2AP is critical for the formation and maintenance of a specialized cell-cell contact between kidney podocyte foot processes, the slit diaphragm. Here, CD2AP links the cell adhesion protein nephrin to the actin cytoskeleton. In addition, CMS/CD2AP binds actin-regulating proteins, such as CAPZ and cortactin, and has been implicated in the internalization of growth factor receptors. We found that CD2AP specifically interacts with the C-terminal domain of Rac1 but not with that of other Rho family members. Efficient interaction between Rac1 and CD2AP requires both the proline-rich domain and the poly-basic region in the Rac1 C terminus, and at least two of the three N-terminal SH3 domains of CD2AP. CD2AP co-localizes with Rac1 to membrane ruffles, and small interfering RNA-based experiments showed that CD2AP links Rac1 to CAPZ and cortactin. Finally, expression of constitutive active Rac1 recruits CD2AP to cell-cell contacts in epithelial cells, where we found CD2AP to participate in the control of the epithelial barrier function. These data identify CD2AP as a novel Rac1-associated adapter protein that participates in the regulation of epithelial cell-cell contact.     

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.     

Side-chain dynamics of the SAP SH2 domain correlate with a binding hot spot and a region with conformational plasticity

X-linked lymphoproliferative disease is caused by mutations in the protein SAP, which consists almost entirely of a single SH2 domain. SAP interacts with the Tyr281 site of the T<-->B cell signaling protein SLAM via its SH2 domain. Interestingly, binding is not dependent on phosphorylation but does involve interactions with residues N-terminal to the Tyr. We have used 15N and 2H NMR relaxation experiments to investigate the motional properties of the SAP SH2 domain backbone amides and side-chain methyl groups in the free protein and complexes with phosphorylated and non-phosphorylated peptides derived from the Tyr281 site of SLAM. The most mobile methyl groups are in side-chains with large RMSD values between the three crystal structures of SAP, suggesting that fast time-scale dynamics in side-chains is associated with conformational plasticity. The backbone amides of two residues which interact with the C-terminal part of the peptides experience fast time-scale motions in the free SH2 domain that are quenched upon binding of either the phosphorylated or non-phosphorylated peptide. Of most importance, the mobility of methyl groups in and around the binding site for residues in the N-terminus of the peptide is significantly restricted in the complexes, underscoring the dominance of this interaction with SAP and demonstrating a correlation between changes in rapid side-chain motion upon binding with local binding energy.     

Solution structure of N-terminal SH3 domain of Vav and the recognition site for Grb2 C-terminal SH3 domain

The three-dimensional structure of the N-terminal SH3 domain (residues 583–660) of murine Vav, which contains a tetra-proline sequence (Pro 607-Pro 610), was determined by NMR. The solution structure of the SH3 domain shows a typical SH3 fold, but it exists in two conformations due to cis-trans isomerization at the Gly614-Pro615 bond. The NMR structure of the P615G mutant, where Pro615 is replaced by glycine, reveals that the tetra-proline region is inserted into the RT-loop and binds to its own SH3 structure. The C-terminal SH3 domain of Grb2 specifically binds to the trans form of the N-terminal SH3 domain of Vav. The surface of Vav N-terminal SH3 which binds to Grb2 C-terminal SH3 was elucidated by chemical shift mapping experiments using NMR. The surface does not involve the tetra-proline region but involves the region comprising the n-src loop, the N-terminal and the C-terminal regions. This surface is located opposite to the tetra-proline containing region, consistent with that of our previous mutagenesis studies.     

Molecular cloning and characterization of a tyrosine phosphatase from Monosiga brevicollis

Protein tyrosine phosphorylation is thought to be a unique feature of multicellular animals. Interestingly, the genome of the unicellular protist Monosiga brevicollis reveals a surprisingly high number and diversity of protein tyrosine kinases, protein tyrosine phosphatases (PTPs), and phosphotyrosine-binding domains. Our study focuses on a hypothetical SH2 domain-containing PTP (SHP), which interestingly has a predicted structure that is distinct from SHPs found in animals. In this study, we isolated cDNA of the enzyme and discovered that its actual sequence was different from the predicted sequence as a result of non-consensus RNA splicing. Contrary to the predicted structure with one SH2 domain and a disrupted phosphatase domain, Monosiga brevicollis SHP (MbSHP) contains two SH2 domains and an intact PTP domain, closely resembling SHP enzymes found in animals. We further expressed the full-length and SH2 domain-truncated forms of the enzyme in Escherichiacoli cells and characterized their enzymatic activities. The double-SH2 domain-truncated form of the enzyme effectively dephosphorylated a common PTP substrate with a specific activity among the highest in characterized PTPs, while the full-length and the N-terminal SH2 domain-truncated forms of the enzyme showed much lower activity with altered pH dependency and responses to ionic strength and common PTP inhibitors. This indicates that SH2 domains suppress the catalytic activity. SHP represents a highly conserved ancient PTP, and studying MbSHP should provide a better understanding about the evolution of tyrosine phosphorylation.     

Dissection of the energetic coupling across the Src SH2 domain-tyrosyl phosphopeptide interface

Src Homology (SH2) domains play critical roles in signaling pathways by binding to phosphotyrosine (pTyr)-containing sequences, thereby recruiting SH2 domain-containing proteins to tyrosine-phosphorylated sites on receptor molecules. Investigations of the peptide binding specificity of the SH2 domain of the Src kinase (Src SH2 domain) have defined the EEI motif C-terminal to the phosphotyrosine as the preferential binding sequence. A subsequent study that probed the importance of eight specificity-determining residues of the Src SH2 domain found two residues which when mutated to Ala had significant effects on binding: Tyr beta D5 and Lys beta D3. The mutation of Lys beta D3 to Ala was particularly intriguing, since a Glu to Ala mutation at the first (+1) position of the EEI motif (the residue interacting with Lys beta D3) did not significantly affect binding. Hence, the interaction between Lys beta D3 and +1 Glu is energetically coupled. This study is focused on the dissection of the energetic coupling observed across the SH2 domain-phosphopeptide interface at and around the +1 position of the peptide. It was found that three residues of the SH2 domain, Lys beta D3, Asp beta C8 and AspCD2 (altogether forming the so-called +1 binding region) contribute to the selection of Glu at the +1 position of the ligand. A double (Asp beta C8Ala, AspCD2Ala) mutant does not exhibit energetic coupling between Lys beta D3 and +1 Glu, and binds to the pYEEI sequence 0.3 kcal/mol tighter than the wild-type Src SH2 domain. These results suggest that Lys beta D3 in the double mutant is now free to interact with the +1 Glu and that the role of Lys beta D3 in the wild-type is to neutralize the acidic patch formed by Asp beta C8 and AspCD2 rather than specifically select for a Glu at the +1 position as it had been hypothesized previously. A triple mutant (Lys beta D3Ala, Asp beta C8Ala, AspCD2Ala) has reduced binding affinity compared to the double (Asp beta C8Ala, AspCD2Ala) mutant, yet binds the pYEEI peptide as well as the wild-type Src SH2 domain. The structural basis for such high affinity interaction was investigated crystallographically by determining the structure of the triple (Lys beta D3Ala, Asp beta C8Ala, AspCD2Ala) mutant bound to the octapeptide PQpYEEIPI (where pY indicates a phosphotyrosine). This structure reveals for the first time contacts between the SH2 domain and the -1 and -2 positions of the peptide (i.e. the two residues N-terminal to pY). Thus, unexpectedly, mutations in the +1 binding region affect binding of other regions of the peptide. Such additional contacts may account for the high affinity interaction of the triple mutant for the pYEEI-containing peptide.     

SETA is a multifunctional adapter protein with three SH3 domains that binds Grb2, Cbl, and the novel SB1 proteins

Expression of the src homology 3 (SH3)-encoding, expressed in tumorigenic astrocytes (SETA) gene is associated with astrocyte transformation in culture and tumors in the adult brain. SETA binds to the apoptosis regulator apoptosis-linked gene 2 (ALG-2) interacting protein 1 (AIP1), and modulates apoptosis in astrocytes. The predicted protein structure of SETA revealed two SH3 domains, while related proteins were reported to have three. Here we report the identification of an additional SH3 domain N-terminal to the previously identified SETA sequence. Yeast two-hybrid screening of a p53(-/-) astrocyte cDNA library with this SH3 domain identified a novel gene, SETA binding protein 1 (SB1), with 55% amino acid identity to the renal tumor antigen, NY-REN-45. In vitro confrontation and co-immunoprecipitation experiments confirmed the binding of SB1 to SETA. Evidence that SETA binds to the CD2 protein, the proto-oncogene c-Cbl, and the signal transduction molecule Grb2, and can dimerize via its C-terminal coiled coil (CC) domain is also presented.     

Crystal structure of the cell corpse engulfment protein CED-2 in Caenorhabditis elegans

In the nematode Caenorhabditis elegans, the cell corpse engulfment proteins CED-2, CED-5, and CED-12 act in the same pathway to regulate the activation of the Rac small GTPase, CED-10, leading to the rearrangement of the actin cytoskeleton for engulfing apoptotic cells. Nevertheless, it is not well understood how these proteins act together. Here we report the crystal structures of the CED-2 protein as determined by X-ray crystallography. The full-length CED-2 protein and its truncated form containing the N-terminal SH2 domain and the first SH3 domain show similar three-dimensional structures. A CED-2 point mutation (F125G) disrupting its interaction with the PXXP motif of CED-5 did not affect its rescuing activity. However, CED-2 was found to interact with the N-terminal region of CED-5. Our findings suggest that CED-2 may regulate cell corpse engulfment by interacting with CED-5 through the N-terminal region rather than the PXXP motif.     

Structural basis for phosphotyrosine recognition by the Src homology-2 domains of the adapter proteins SH2-B and APS

SH2-B, APS, and Lnk constitute a family of adapter proteins that modulate signaling by protein tyrosine kinases. These adapters contain an N-terminal dimerization region, a pleckstrin homology domain, and a C-terminal Src homology-2 (SH2) domain. SH2-B is recruited via its SH2 domain to various protein tyrosine kinases, including Janus kinase-2 (Jak2) and the insulin receptor. Here, we present the crystal structure at 2.35 A resolution of the SH2 domain of SH2-B in complex with a phosphopeptide representing the SH2-B recruitment site in Jak2 (pTyr813). The structure reveals a canonical SH2 domain-phosphopeptide binding mode, but with specific recognition of a glutamate at the +1 position relative to phosphotyrosine, in addition to recognition of a hydrophobic residue at the +3 position. Biochemical studies of SH2-B and APS demonstrate that, although the SH2 domains of these two adapter proteins share 79% sequence identity, the SH2-B SH2 domain binds preferentially to Jak2, whereas the APS SH2 domain has higher affinity for the insulin receptor. This differential specificity is attributable to the difference in the oligomeric states of the two SH2 domains: monomeric for SH2-B and dimeric for APS.     

Phosphopeptide binding to the N-terminal SH2 domain of the p85 alpha subunit of PI 3'-kinase: a heteronuclear NMR study.

The N-terminal src-homology 2 domain of the p85 alpha subunit of phosphatidylinositol 3' kinase (SH2-N) binds specifically to phosphotyrosine-containing sequences. Notably, it recognizes phosphorylated Tyr 751 within the kinase insert of the cytoplasmic domain of the activated beta PDGF receptor. A titration of a synthetic 12-residue phosphopeptide (ESVDY*VPMLDMK) into a solution of the SH2-N domain was monitored using heteronuclear 2D and 3D NMR spectroscopy. 2D-(15N-1H) heteronuclear single-quantum correlation (HSQC) experiments were performed at each point of the titration to follow changes in both 15N and 1H chemical shifts in NH groups. When mapped onto the solution structure of the SH2-N domain, these changes indicate a peptide-binding surface on the protein. Line shape analysis of 1D profiles of individual (15N-1H)-HSQC peaks at each point of the titration suggests a kinetic exchange model involving at least 2 steps. To characterize changes in the internal dynamics of the domain, the magnitude of the (15N-1H) heteronuclear NOE for the backbone amide of each residue was determined for the SH2-N domain with and without bound peptide. These data indicate that, on a nanosecond timescale, there is no significant change in the mobility of either loops or regions of secondary structure. A mode of peptide binding that involves little conformational change except in the residues directly involved in the 2 binding pockets of the p85 alpha SH2-N domain is suggested by this study.     

The SH2B1 adaptor protein associates with a proximal region of the erythropoietin receptor

Gene targeting experiments have shown that the cytokine erythropoietin (EPO), its cognate erythropoietin receptor (EPO-R), and associated Janus tyrosine kinase, JAK2, are all essential for erythropoiesis. Structural-functional and murine knock-in experiments have suggested that EPO-R Tyr-343 is important in EPO-mediated mitogenesis. Although Stat5 binds to EPO-R phosphotyrosine 343, the initial Stat5-deficient mice did not have profound erythroid abnormalities suggesting that additional Src homology 2 (SH2) domain-containing effectors may bind to EPO-R Tyr-343 and couple to downstream signaling pathways. We have utilized cloning of ligand target (COLT) screening to demonstrate that EPO-R Tyr(P)-343 and Tyr(P)-401 bind to the SH2 domain-containing adaptor protein SH2B1β. Immunoprecipitation and in vitro mixing experiments reveal that EPO-R binds to SH2B1 in an SH2 domain-dependent manner and that the sequence that confers SH2B1 binding to the EPO-R is pYXXL. Previous studies have shown that SH2B1 binds directly to JAK2, but we show that in hematopoietic cells, SH2B1β preferentially associates with the EPO-R. SH2B1 is capable of constitutive association with EPO-R, which is necessary for its optimal SH2-dependent recruitment to EPO-R-Tyr(P)-343/Tyr(P)-401. We also demonstrate that SH2B1 is responsive to EPO stimulation and becomes phosphorylated, most likely on serines/threonines, in an EPO dose- and time-dependent manner. In the absence of SH2B1, we observe enhanced activation of signaling pathways downstream of the EPO-R, indicating that SH2B1 is a negative regulator of EPO signaling.     

人EEN蛋白的结构预测和功能分析

采用基于神经网络的算法预测了我们自行克隆的新的白血病相关蛋白ＥＥＮ（ｅｘｔｒａ ｅｌｅｖｅｎｎｉｎｅｔｅｅｎ, ＥＥＮ）全长分子的二级结构,结果表明：ＥＥＮ 蛋白可能有三个结构域,Ｎ 端由三段α螺旋和短β折叠组成,中间为四段α螺旋组成的四螺旋结构,Ｃ端为ＳＨ３结构域,类似于在受体酪氨酸激酶信号传导途径中起重要作用的ＳＥＭ－５／ＧＲＢ２ Ｃ端ＳＨ３结构域;利用同源蛋白结构模拟的方法,模拟了ＥＥＮ ＳＨ３结构域的三维结构,结果表明：ＥＥＮ ＳＨ３结构域与ＳＥＭ－５／ＧＲＢ２ ＳＨ３结构域具有相近的结构,构成脯氨酸结合区的氨基酸非常保守．上述结果提示：ＥＥＮ 蛋白可能为新的信号蛋白,可能涉及新的信号传导途径或新的信号传导旁路,ＳＨ３结构域是其功能区域．     

POSH2 is a RING finger E3 ligase with Rac1 binding activity through a partial CRIB domain

The Plenty of SH3 domains protein (POSH) is an E3 ligase and a scaffold in the JNK mediated apoptosis, linking Rac1 to downstream components.We here describe POSH2 which was identified from a p21-activated kinase 2 (PAK2) interactor screen. POSH2 is highly homologous with other members of the POSH family; it contains four Src homology 3 (SH3) domains and a RING finger domain which confers E3 ligase activity to the protein. In addition POSH2 contains an N-terminal extension which is conserved among its mammalian counterparts. POSH2 interacts with GTP-loaded Rac1. We have mapped this interaction to a previously unrecognized partial Cdc42/Rac1-interactive binding domain.

Structured summary

MINT-7987761: POSH1 (uniprotkb:Q9HAM2) physically interacts (MI:0915) with Ubiquitin (uniprotkb:P62988) by anti tag coimmunoprecipitation (MI:0007)MINT-7987932: PAK2 (uniprotkb:Q13177) binds (MI:0407) to CDC42 (uniprotkb:Q07912) by solid phase assay (MI:0892)MINT-7987908: POSH1 (uniprotkb:Q9HAM2) binds (MI:0407) to Rac1 (uniprotkb:P63000) by solid phase assay (MI:0892)MINT-7987880: POSH2 (uniprotkb:Q8TEJ3) binds (MI:0407) to Rac1 (uniprotkb:P63000) by solid phase assay (MI:0892)MINT-7987734: POSH2 (uniprotkb:Q8TEJ3) physically interacts (MI:0915) with Ubiquitin (uniprotkb:P62988) by anti tag coimmunoprecipitation (MI:0007)MINT-7987779, MINT-7987804, MINT-7987824, MINT-7987838, MINT-7987853: Rac1 (uniprotkb:P63000) physically interacts (MI:0915) with POSH2 (uniprotkb:Q8TEJ3) by anti tag coimmunoprecipitation (MI:0007)MINT-7987920: PAK2 (uniprotkb:Q13177) binds (MI:0407) to Rac1 (uniprotkb:P63000) by solid phase assay (MI:0892)     

P120-Ras GTPase activating protein (RasGAP): A multi-interacting protein in downstream signaling

p120-RasGAP (Ras GTPase activating protein) plays a key role in the regulation of Ras-GTP bound by promoting GTP hydrolysis via its C-terminal catalytic domain. The p120-RasGAP N-terminal part contains two SH2, SH3, PH (pleckstrin homology) and CaLB/C2 (calcium-dependent phospholipid-binding domain) domains. These protein domains allow various functions, such as anti-/pro-apoptosis, proliferation and also cell migration depending of their distinct partners. The p120-RasGAP domain participates in protein–protein interactions with Akt, Aurora or RhoGAP to regulate functions described bellow. Here, we summarize, in angiogenesis and cancer, the various functional roles played by p120-RasGAP domains and their effector partners in downstream signaling.     

Hydrophobic Interaction between the SH2 Domain and the Kinase Domain Is Required for the Activation of Csk

The protein tyrosine kinase C-terminal Src kinase (Csk) is activated by the engagement of its Src homology (SH) 2 domain. However, the molecular mechanism required for this is not completely understood. The crystal structure of the active Csk indicates that Csk could be activated by contact between the SH2 domain and the β3-αC loop in the N-terminal lobe of the kinase domain. To study the importance of this interaction for the SH2-domain-mediated activation of Csk, we mutated the amino acid residues forming the contacts between the SH2 domain and the β3-αC loop. The mutation of the β3-αC loop Ala228 to glycine and of the SH2 domain Tyr116, Tyr133, Leu138, and Leu149 to alanine resulted in the inability of the SH2 domain ligand to activate Csk. Furthermore, the overexpressed Csk mutants A228G, Y133A/Y116A, L138A, and L149A were unable to efficiently inactivate endogenous Src in human embryonic kidney 293 cells. The results suggest that the SH2-domain-mediated activation of Csk is dependent on the binding of the β3-αC loop Ala228 to the hydrophobic pocket formed by the side chains of Tyr116, Tyr133, Leu138, and Leu149 on the surface of the SH2 domain.     

A yeast 2-hybrid analysis of human GTP cyclohydrolase I protein interactions

The yeast 2-hybrid system was used to identify protein domains involved in the oligomerization of human guanosine 5'-triphosphate (GTP) Cyclohydrolase I (GCH1) and the interaction of GCH1 with its regulatory partner, GCH1 feedback regulatory protein (GFRP). When interpreted within the structural framework derived from crystallography, our results indicate that the GCH1 N-terminal alpha-helices are not the only domains involved in the formation of dimers from monomers and also suggest an important role for the C-terminal alpha-helix in the assembly of dimers to form decamers. Moreover, a previously unknown role of the extended N-terminal alpha-helix in the interaction of GCH1 and GFRP was revealed. To discover novel GCH1 protein binding partners, we used the yeast 2-hybrid system to screen a human brain library with GCH1 N-terminal amino acids 1-96 as prey. This protruding extension of GCH1 contains two canonical Type-I Src homology-3 (SH3) ligand domains located within amino acids 1-42. Our screen yielded seven unique clones that were subsequently shown to require amino acids 1-42 for binding to GCH1. The interaction of one of these clones, Activator of Heat Shock 90 kDa Protein (Aha1), with GCH1 was validated by glutathione-s-transferase (GST) pull-down assay. Although the physiological relevance of the Aha1-GCH1 interaction requires further study, Aha1 may recruit GCH1 into the endothelial nitric oxide synthase/heat shock protein (eNOS/Hsp90) complex to support changes in endothelial nitric oxide production through the local synthesis of BH4.     

Tight association of GRB2 with receptor protein-tyrosine phosphatase alpha is mediated by the SH2 and C-terminal SH3 domains.

Receptor protein-tyrosine phosphatase alpha (RPTPalpha), a transmembrane member of the extensive family of protein-tyrosine phosphatases (PTPs), is constitutively phosphorylated on Tyr789, a consensus binding site for the SH2 domain of the SH3-SH2-SH3 adaptor protein GRB2. We have previously shown that GRB2 binds to P.Tyr789 in vivo and in vitro via its SH2 domain. Here, we report that not only the GRB2 SH2 domain, but also the C-terminal SH3 domain is involved in binding to RPTPalpha in vitro and in vivo. Although the N-terminal SH3 domain of GRB2 is essential for binding to the Ras guanine nucleotide exchange factor Son of Sevenless (Sos), an RPTPalpha-GRB2-Sos complex could not be detected. The inclusion of peptides encompassing an hSos1 proline-rich motif in cell lysates resulted in enhanced binding of RPTPalpha to GRB2 in vitro, suggesting that steric hindrance prohibits formation of the RPTPalpha-GRB2-Sos complex. In vitro binding experiments indicated that the binding of GRB2 to Sos/dynamin and RPTPalpha was mutually exclusive. Analysis of in vitro binding kinetics coupled with results from transient co-transfections demonstrated that RPTPalpha is tightly bound to GRB2. The site of interaction of the C-terminal SH3 domain of GRB2 with RPTPalpha was mapped using deletion mutants to an 18-residue region in the N-terminal PTP domain. Arg469, within this region, was identified as one of the residues that is involved in the interaction with the C-terminal SH3 domain of GRB2. RPTPalpha residues 469-486 are localized close to the catalytic site cleft in the structure of the N-terminal PTP-domain, suggesting that interaction with the C-terminal SH3 domain may block access to the catalytic site, thus inhibiting RPTPalpha activity.     

Protein kinase cθ c2 domain is a phosphotyrosine binding module that plays a key role in its activation

Protein kinase Cθ (PKCθ) is a novel PKC that plays a key role in T lymphocyte activation. To understand how PKCθ is regulated in T cells, we investigated the properties of its N-terminal C2 domain that functions as an autoinhibitory domain. Our measurements show that a Tyr(P)-containing peptide derived from CDCP1 binds the C2 domain of PKCθ with high affinity and activates the enzyme activity of the intact protein. The Tyr(P) peptide also binds the C2 domain of PKCδ tightly, but no enzyme activation was observed with PKCδ. Mutations of PKCθ-C2 residues involved in Tyr(P) binding abrogated the enzyme activation and association of PKCθ with Tyr-phosphorylated full-length CDCP1 and severely inhibited the T cell receptor/CD28-mediated activation of a PKCθ-dependent reporter gene in T cells. Collectively, these studies establish the C2 domain of PKCθ as a Tyr(P)-binding domain and suggest that the domain may play a major role in PKCθ activation via its Tyr(P) binding.     

Two phosphorylation-independent sites on the p85 SH2 domains bind A-Raf kinase.

Src homology 2 (SH2) domains mediate phosphotyrosine (pY)-dependent protein:protein interactions involved in signal transduction pathways. We have found that the SH2 domains of the 85-kDa alpha subunit (p85) of phosphatidylinositol 3-kinase (PI3 kinase) bind directly to the serine/threonine kinase A-Raf. In this report we show that the p85 SH2:A-Raf interaction is phosphorylation-independent. The affinity of the p85 C-SH2 domain for A-Raf and phosphopeptide pY751 was similar, raising the possibility that a p85:A-Raf complex may play a role in the coordinated regulation of the PI3 kinase and Raf-MAP kinase pathways. We further show that the p85 C-SH2 domain contains two distinct binding sites for A-Raf; one overlapping the phosphotyrosine-dependent binding site and the other a separate phosphorylation-independent site. This is the first evidence for a second binding site on an SH2 domain, distinct from the phosphotyrosine-binding pocket.