SH3 Binding Research Articles

Structural basis of the differential binding of the SH3 domains of Grb2 adaptor to the guanine nucleotide exchange factor Sos1

Grb2–Sos1 interaction, mediated by the canonical binding of N-terminal SH3 (nSH3) and C-terminal SH3 (cSH3) domains of Grb2 to a proline-rich sequence in Sos1, provides a key regulatory switch that relays signaling from activated receptor tyrosine kinases to downstream effector molecules such as Ras. Here, using isothermal titration calorimetry in combination with site-directed mutagenesis, we show that the nSH3 domain binds to a Sos1-derived peptide containing the proline-rich consensus motif PPVPPR with an affinity that is nearly threefold greater than that observed for the binding of cSH3 domain. We further demonstrate that such differential binding of nSH3 domain relative to the cSH3 domain is largely due to the requirement of a specific acidic residue in the RT loop of the β-barrel fold to engage in the formation of a salt bridge with the arginine residue in the consensus motif PPVPPR. While this role is fulfilled by an optimally positioned D15 in the nSH3 domain, the chemically distinct and structurally non-equivalent E171 substitutes in the case of the cSH3 domain. Additionally, our data suggest that salt tightly modulates the binding of both SH3 domains to Sos1 in a thermodynamically distinct manner. Our data further reveal that, while binding of both SH3 domains to Sos1 is under enthalpic control, the nSH3 binding suffers from entropic penalty in contrast to entropic gain accompanying the binding of cSH3, implying that the two domains employ differential thermodynamic mechanisms for Sos1 recognition. Our new findings are rationalized in the context of 3D structural models of SH3 domains in complex with the Sos1 peptide. Taken together, our study provides structural basis of the differential binding of SH3 domains of Grb2 to Sos1 and a detailed thermodynamic profile of this key protein–protein interaction pertinent to cellular signaling and cancer.

Tyrosine Phosphorylation in the SH3 Domain Disrupts Negative Regulatory Interactions within the c-Abl Kinase Core

Recent studies have shown that trans-phosphorylation of the Abl SH3 domain at Tyr89 by Src-family kinases is required for the full transforming activity of Bcr-Abl. Tyr89 localizes to a binding surface of the SH3 domain that engages the SH2-kinase linker in the crystal structure of the c-Abl core. Displacement of SH3 from the linker is likely to influence efficient downregulation of c-Abl. Hydrogen-deuterium exchange (HX) and mass spectrometry (MS) were used to investigate whether Tyr89 phosphorylation affects the ability of the SH3 domain to interact intramolecularly with the SH2-kinase linker in cis as well as other peptide ligands in trans. HX MS analysis of SH3 binding showed that when various Abl constructs were phosphorylated at Tyr89 by the Src-family kinase Hck, SH3 was unable to engage a high-affinity ligand in trans and that interaction with the linker in cis was reduced dramatically in a construct containing the SH3 and SH2 domains plus the linker. Phosphorylation of the Abl SH3 domain on Tyr89 also interfered with binding to the negative regulatory protein Abi-1 in trans. Site-directed mutagenesis of Tyr89 and Tyr245, another tyrosine phosphorylation site located in the linker that may also influence SH3 binding, implicated Tyr89 as the key residue necessary for disrupting regulation after phosphorylation. These results imply that phosphorylation at Tyr89 by Src-family kinases prevents engagement of the Abl SH3 domain with its intramolecular binding partner leading to enhanced Abl kinase activity and cellular signaling.

Mechanism of Influenza A Virus NS1 Protein Interaction with the p85β, but Not the p85α, Subunit of Phosphatidylinositol 3-Kinase (PI3K) and Up-regulation of PI3K Activity

Influenza A virus infection activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway by binding influenza A virus NS1 protein to the p85beta regulatory subunit of PI3K. In this study, we report that NS1 binds to the inter-SH2 (iSH2) domain of p85beta. Mutational analyses on p85beta iSH2 domain defined that Val-573 is the critical amino acid (AA) that mediates NS1 and p85beta interaction. In reciprocal gain of function experiments with p85alpha, we demonstrated that mutation to Val at Met-582 leads to NS1 binding and increased PI3K activity. Molecular modeling based on our experimental results suggested that, in addition to the interaction interface between the NS1 SH3 binding motif 1 (AA 164-167) and p85beta Val-573, AA 137-142 in NS1 might interact with p85beta. Indeed, mutations of AA 141 and 142 in NS1 disrupted the interaction between NS1 and p85beta. Mutant virus PR8-NS1-141/142 was not able to activate Akt phosphorylation. Furthermore, PI3K assays demonstrated that, in wild-type virus-infected cells, p85beta-associated PI3K activity was increased significantly. In contrast, in the mutant virus-infected cells containing mutant NS1 unable to interact with p85beta, the p85beta-associated PI3K activity up-regulation was not seen, suggesting that PI3K up-regulation is dependent upon the interaction between NS1 and p85beta. Competition experiments and the immunoprecipitation studies demonstrated that NS1, p85beta, and p110 form a complex in cells. Finally, the mechanism by which binding of NS1 to p85beta regulates PI3K activity was discussed based on a predicted structural model of NS1-p85-p110 complex.

Phosphorylation Regulates Tau Interactions with Src Homology 3 Domains of Phosphatidylinositol 3-Kinase, Phospholipase Cγ1, Grb2, and Src Family Kinases

The microtubule-associated protein tau can associate with various other proteins in addition to tubulin, including the SH3 domains of Src family tyrosine kinases. Tau is well known to aggregate to form hyperphosphorylated filamentous deposits in several neurodegenerative diseases (tauopathies) including Alzheimer disease. We now report that tau can bind to SH3 domains derived from the p85alpha subunit of phosphatidylinositol 3-kinase, phospholipase Cgamma1, and the N-terminal (but not the C-terminal) SH3 of Grb2 as well as to the kinases Fyn, cSrc, and Fgr. However, the short inserts found in neuron-specific isoforms of Src prevented the binding of tau. The experimentally determined binding of tau peptides is well accounted for when modeled into the peptide binding cleft in the SH3 domain of Fyn. After phosphorylation in vitro or in transfected cells, tau showed reduced binding to SH3 domains; no binding was detected with hyperphosphorylated tau isolated from Alzheimer brain, but SH3 binding was restored by phosphatase treatment. Tau mutants with serines and threonines replaced by glutamate, to mimic phosphorylation, showed reduced SH3 binding. These results strongly suggest that tau has a potential role in cell signaling in addition to its accepted role in cytoskeletal assembly, with regulation by phosphorylation that may be disrupted in the tauopathies including Alzheimer disease.

Proline rich regions of coenzyme A synthase α and β interact with SH3 domains of signaling proteins in vitro

© O. S. BREUS, G. G. PANASYUK, I. T. GOUT, V. V. FILONENKO, I. O. NEMAZANYY, 2008.Coenzyme A-synthases α and β (CoASy α and CoASy β) contain proline rich regions which may bring them into complexes with SH3-domain containing proteins. To test whether CoASy isoforms can bind to SH3 domains we performed in vitro pull down experiments. It was found that CoASy β N-terminal extension, which is especially abundant in prolines, can interact specifically and directly with SH3 domains of tyrosine kinases Fyn and CSK, phospholipase Cγ, NADPH oxidase activator 1 - p67phox, and cytoskeleton protein spectrin. Furthermore, C-terminal SH3 domain of p67phox can also interact with SH3 binding site that resides on the shared part of CoASy β and CoASy α These data demonstrated that CoA Synthases could be involved in complexes with signaling proteins in living cells which may regulate enzymatic activities of CoA Synthases or vice versa CoA Synthase may modulate some steps in signal transduction in the cell in currently unknown way.

Regulation of ubiquitinated protein sorting at multivesicular endosomes

Ubiquitinated membrane proteins are transported into the interior of the lysosome for degradation. This sorting pathway involves incorporation of cargo proteins into endosomal intralumenal vesicles, which accumulate to form a multivesicular endosome/body (MVB). The apparatus that recognizes ubiquitinated membrane cargo proteins in yeast includes Vps27 and Hse1, which are orthologous to Hrs and STAM in animal cells. We find that the Vps27‐Hse1 ubiquitin‐sorting complex associates with a variety of factors that help regulate the fate of ubiquitinated cargo. Recently, we discovered that Ufd3/Doa1 directly binds the SH3 domain of Hse1. Doa1 associates with the AAA ATPase Cdc48, which is involved in processing a number of ubiquitinated substrates. We find that Doa1 possesses 3 distinct ubiquitin binding domains, and a novel SH3 ligand motif required for Hse1 binding. We also found that Doa1 can partially localize to endosomes. Unlike doa1 null mutants, Doa1 mutants lacking the SH3 binding motif for Hse1 have normal levels of ubiquitin and display no growth defects; however, these Doa1 mutants are defective in sorting particular MVB cargo proteins. We also find that the novel Cdc48 cofactor Ubx3 also plays a role at the endosome similar to Doa1 and that ubx3 null mutants are defective in aspects of MVB sorting. These results point to a novel role for a Cdc48‐Doa1‐Ubx3 complex on endosomes that may foster proper processing of ubiquitinated MVB cargo.

Allosteric inhibition of the nonMyristoylated c-Abl tyrosine kinase by phosphopeptides derived from Abi1/Hssh3bp1

Here we report c-Abl kinase inhibition mediated by a phosphotyrosine located in trans in the c-Abl substrate, Abi1. The mechanism, which is pertinent to the nonmyristoylated c-Abl kinase, involves high affinity concurrent binding of the phosphotyrosine pY213 to the Abl SH2 domain and binding of a proximal PXXP motif to the Abl SH3 domain. Abi1 regulation of c-Abl in vivo appears to play a critical role, as demonstrated by inhibition of pY412 phosphorylation of the nonmyristoylated Abl by coexpression of Abi1. Pervanadate-induced c-Abl kinase activity was also reduced upon expression of the wild type Abi1 but not by expression of the Y213 to F213 mutant Abi1 in LNCaP cells, which are naturally deficient in the regulatory pY213. Our findings suggest a novel mechanism by which Abl kinase is regulated in cells.

Binding of the Proline-Rich Segment of Myelin Basic Protein to SH3 Domains: Spectroscopic, Microarray, and Modeling Studies of Ligand Conformation and Effects of Posttranslational Modifications

Myelin basic protein (MBP) is a multifunctional protein involved in maintaining the stability and integrity of the myelin sheath by a variety of interactions with membranes and with cytoskeletal and other proteins. A central segment of MBP is highly conserved in mammals and consists of a membrane surface-associated amphipathic alpha-helix, immediately followed by a proline-rich segment that we hypothesize is an SH3 ligand. We show by circular dichroic spectroscopy that this proline-rich segment forms a polyproline type II helix in vitro under physiological conditions and that phosphorylation at a constituent threonyl residue has a stabilizing effect on its conformation. Using SH3 domain microarrays, we observe that the unmodified recombinant murine 18.5 kDa MBP isoform (rmC1 component) binds the following SH3 domains: Yes1 > PSD95 > cortactin = PexD = Abl = Fyn = c-Src = Itk in order of decreasing affinity. A quasi-deiminated form of the protein (rmC8) binds the SH3 domains Yes1 > Fyn > cortactin = c-Src > PexD = Abl. Phosphorylation of rmC1 at 1-2 threonines within the proline-rich segment by mitogen-activated protein kinase in vitro has no effect on the binding specificity to the SH3 domains on the array. An SH3 domain of chicken Fyn is also demonstrated to bind to lipid membrane-associated C1, phosphorylated C1, and rmC8. Molecular docking simulations of the interaction of the putative SH3 ligand of classic MBP with the human Fyn SH3 domain indicate that the strength of the interaction is of the same order of magnitude as with calmodulin and that the molecular recognition and association is mediated by some weak CH...pi interactions between the ligand prolyl residues and the aromatic ones of the SH3 binding site. One such interaction is well-conserved and involves the stacking of an MBP-peptide prolyl and an SH3 domain tryptophanyl residue, as in most other SH3-ligand complexes. Lysyl and arginyl residues in the peptide canonically interact via salt bridges and cation-pi interactions with negatively charged and aromatic residues in the SH3 domain binding site. Posttranslational modifications (phosphorylation or methylation) of the ligand cause noticeable shifts in the conformation of the flexible peptide and its side chains but do not predict any major inhibition of the binding beyond somewhat less favorable interactions for peptides with phosphorylated seryl or threonyl residues.

Protein protein interaction inhibition (2P2I) combining high throughput and virtual screening: Application to the HIV-1 Nef protein.

Protein-protein recognition is the cornerstone of multiple cellular and pathological functions. Therefore, protein-protein interaction inhibition (2P2I) is endowed with great therapeutic potential despite the initial belief that 2P2I was refractory to small-molecule intervention. Improved knowledge of complex molecular binding surfaces has recently stimulated renewed interest for 2P2I, especially after identification of "hot spots" and first inhibitory compounds. However, the combination of target complexity and lack of starting compound has thwarted experimental results and created intellectual barriers. Here we combined virtual and experimental screening when no previously known inhibitors can be used as starting point in a structure-based research program that targets an SH3 binding surface of the HIV type I Nef protein. High-throughput docking and application of a pharmacophoric filter on one hand and search for analogy on the other hand identified drug-like compounds that were further confirmed to bind Nef in the micromolar range (isothermal titration calorimetry), to target the Nef SH3 binding surface (NMR experiments), and to efficiently compete for Nef-SH3 interactions (cell-based assay, GST pull-down). Initial identification of these compounds by virtual screening was validated by screening of the very same library of compounds in the cell-based assay, demonstrating that a significant enrichment factor was attained by the in silico screening. To our knowledge, our results identify the first set of drug-like compounds that functionally target the HIV-1 Nef SH3 binding surface and provide the basis for a powerful discovery process that should help to speed up 2P2I strategies and open avenues for new class of antiviral molecules.

Rac1 GTPase is activated by hepatitis B virus replication — involvement of HBX

Hepatitis B virus (HBV) is a causative agent for liver diseases including hepatocellular carcinoma. Understanding its interactions with cellular proteins is critical in the elucidation of the mechanisms of disease progression. Using a cell-based HBV replication system, we showed that HBV replication in HepG2 cells resulted in a cellular morphological changes displaying membrane rufflings and lamellipodia like structures reminiscent of cells expressing constitutively activated Rac1. We also showed that activated Rac1 resulted in increased viral replication. HBV replication specifically activated wild type Rac1, but not Cdc42. The Rac1 activation by HBV replication also resulted in the phosphorylation of ERK1/2 and AKT, the downstream targets of Rac1 signaling cascade. The smallest HBV viral protein, HBX, was able to activate the endogenous Rac1 and induce membrane ruffling when transfected into cells. Significantly, HBX was found to directly interact with a Rac1 nucleotide exchange factor (βPIX) through a SH3 binding motif. Taken together, we have shown the interaction of HBV with the Rho GTPase, affecting cell morphology through the Rac1 activation pathway. HBV may possibly make use of an activated Rac1 signaling pathway for increased replication and resultant metastatic effects.

Dissection of Bcr-abl Structural Domains Relating to Kinase Auto-Inhibition Using a Forward Mutational Screen with the Non-ATP Competitive Inhibitor GNF-2.

The Bcr-abl kinase is the causative agent for chronic myeloid leukemia (CML) and has been established as the primary clinical target for treatment of the disease through extensive use of Imatinib. Imatinib is the defining member of a class of ATP-binding site competitive inhibitors that lock Bcr-abl in an inactive conformation. Mutational screens of Bcr-abl using Imatinib and its derivatives as probes have been highly informative in prediction of clinically relevant mutations of Bcr-abl as well as in revealing the structure/function relationship of the kinase in general. Using compounds with a distinct mechanism of action from the Imatinib class to interrogate Bcr-abl will contribute to both more complete understanding of kinase function as well as to potential combination therapies for more effective treatment of CML. GNF-2, a recently identified inhibitor of Bcr-abl, establishes a new class of non-ATP competitive Bcr-abl family kinase inhibitors that may be developed as therapeutic agents for CML. GNF-2 effectively impairs the in vivo kinase activity of Bcr-abl and the growth of Bcr-abl transformed cells. GNF-2 functions at least in part through association with the myristate binding pocket of Bcr-abl. In order to further elucidate the mechanism of GNF-2 action as well as clinically relevant GNF-2 resistant mutants of Bcr-abl, a mutational screen coupling Bcr-abl mutagenesis to selection of drug resistance was performed using GNF-2 as probe. A number of functionally distinct resistant Bcr-abl mutations were recovered. Over half of all GNF-2 resistant clones harbored Bcr-abl mutations affecting the myristate binding pocket or the abl-SH3 domain, suggesting two potential methods of mutational resistance.1.The myristate binding domain mutants support a direct resistance model whereby GNF-2 association with Bcr-abl is impaired by disruption of the myristate binding pocket.2.Given a previous report that GNF-2 cannot inhibit Bcr-abl kinase activity in vitro, a novel model emerges for indirect resistance to GNF-2 by SH3 mutants that lose affinity for an inhibitory associated protein.The indirect resistance model specifically suggests that GNF-2 association confers a structural state of wildtype Bcr-abl which facilitates association to a putative inhibitory binding partner, thereby affecting inhibition. Indeed, the strongest of several candidate inhibitory binding partners, the Abl-SH3 domain binding inhibitor Abi-2 was observed to co-immunoprecipitate with Bcr-abl in the presence of GNF-2. This association correlated with reduced Bcr-abl auto-phosphorylation levels. These observations provide preliminary support for an indirect mechanism of Bcr-abl inhibition by GNF-2. Additional experiments involving shRNA knockdown of Abi-2 are being completed to determine the requirement of this Bcr-abl binding partner for GNF-2 activity. Further characterization of the SH3 and myristate binding domain mutants in the context of Abi-2 and GNF-2 binding affinities may establish a previously undescribed indirect mechanism of Bcr-abl inhibition by an allosteric non-ATP inhibitor.

Microarray Identification of Differentially Expressed Genes in AHI-1- Mediated Leukemic Transformation of Human Cutaneous T-Cell Lymphoma Cells.

Abelson helper integration site 1 (Ahi-1) is a recently identified oncogene which is often the target of provirus insertional mutagenesis in murine leukemias and lymphomas. Ahi-1/AHI-1 encodes a unique cell signaling protein with a SH3 domain, multiple SH3 binding sites, seven WD40 repeats and a number of potential tyrosine kinase phosphorylation sites. Its involvement in human leukemogenesis is demonstrated by gross perturbations in its expression in several leukemic cells lines, particularly in cutaneous T-cell lymphoma (CTCL) cell lines (Hut 78 and Hut 102) where 40-fold up-regulation of AHI-1 transcripts is seen. Hut78 cells are derived from a patient with Sezary syndrome, a common leukemic variant of human CTCL. Interestingly, we have recently demonstrated that aberrant expression of AHI-1 at both RNA and protein levels is found in CD4+CD7− leukemic Sezary cells from patients with Sezary syndrome. Moreover, stable suppression of AHI-1 using retroviral-mediated RNA interference in Hut 78 cells reduces autocrine production of interleukin (IL)-2, IL-4 and tumor necrosis factor-alpha (TNFα) and normalizes their transforming activity both in vitro and in vivo. In an effort to identify genes involved in AHI-1-mediated leukemic transformation in CTCL, Microarray analysis was performed using the Affymetrix Human Genome U133 plus 2.0 Arrays which contains over 47,000 transcripts (54,330 probes). Comparative analysis of six RNA samples from AHI-1/sh4 cells (knockdown of AHI-1) and five samples from Hut 78 and Hut 78 cells transduced with a control vector (RPG) demonstrated that 101 genes (119 probes) were statistically differentially expressed with fold changes > or < 2 at p-values ≤ 0.001, using a DNA-Chip Analyzer (dChip). With the same selection criteria, the Linear Model for Microarray Data (Limma) analysis initially listed 239 genes (283 probes). Once the p-values for these genes were adjusted, using the approach of Benjamini and Hochberg, the list was refined to 27 genes (33 probes). After evaluation of differentially expressed genes selected by both dChip and Limma analyses, 15 up-regulated genes (fold change range: 2.2–11) and 6 down-regulated genes (fold change range: 2.1–8.3) in AHI-1 suppressed Hut 78 cells as compared to control cells, were chosen for further analysis. The expression patterns of these 21 genes identified by Microarray analyses were confirmed by quantitative real-time RT-PCR (fold change range: 6.7 higher to 33.3 lower, p≤0.001). Significant down-regulation of AHI-1 itself (p≤0.001) was confirmed in all six AHI-1/sh4 RNA samples studied. Interestingly, functional grouping of differentially expressed genes in AHI-1/sh4 cells shows several are involved in signal transduction (BRDG1, HCK, and REPS2), cell cycle control (CCNG2, CDKN1C, and PDCD6), cell proliferation and differentiation (BIN1 and MLLT11) and mRNA stability (ELAVL1). Additionally, our observed deregulated expression of NKG7 has also recently been documented in both Sezary syndrome and mycosis fungoides patient samples. Further, candidate tumor suppressor genes (BIN1 and CDKN1C) were found to be highly elevated in AHI-1/sh4 cells where AHI-1 expression is stably inhibited. These findings indicate that we have identified several new differentially expressed genes that may play critical roles in alteration of T-cell signaling in AHI-1 mediated leukemic transformation of human CTCL cells.

Interaction of a Novel Signaling Protein (AHI-1) with BCR-ABL Modulates BCR-ABL Transforming Activity and Imatinib Response of Chronic Myeloid Leukemia Progenitor Cells.

Ahi-1 (Abelson helper integration site 1) is a novel gene that was initially identified by provirus insertional mutagenesis in v-abl-induced murine pre-B cell lymphoma as a candidate cooperate oncogene. The Ahi-1 protein has a SH3 domain, multiple SH3 binding sites and WD-repeat domains, suggesting novel signaling activities. We recently demonstrated that Ahi-1/AHI-1 transcript levels are highly deregulated in human leukemic cells, particularly in highly enriched populations of BCR-ABL+ leukemic stem cells from patients with chronic myeloid leukemia (CML) where levels of BCR-ABL transcripts are also elevated. Interestingly, overexpression of Ahi-1 alone in primitive murine hematopotic cells confers a proliferative advantage in vitro and induces a lethal leukemia in vivo; these effects can be enhanced by BCR-ABL, a fusion oncogene that drives CML pathogenesis. To investigate whether deregulated expression of AHI-1 may directly contribute to BCR-ABL-mediated transformation and altered signaling pathways in human CML, knockdown of AHI-1 expression in K562 cells, a cell line that was derived from a patient with CML and that is characterized by highly increased expression of AHI-1, was performed using retroviral-mediated RNA interference. Retroviral-mediated suppression specifically inhibited endogenous AHI-1 expression in transduced cells by 70% as evaluated by Q-RT-PCR and Western blot analyses. It further caused a significant reduction in their growth factor independence in semi-solid cultures (up to 5-fold, P< 0.01) and in single cell cultures (2-fold, P< 0.05) by comparison to cells transduced with a control vector. Importantly, lentiviral-mediated suppression of AHI-1 in BCR-ABL transduced primitive human cord blood (CB) cells (lin−CD34+) further showed a significant reduction of up to 10-fold (P< 0.01) in colony-forming cell (CFC) output and up to 5-fold (P< 0.01) in liquid suspension cultures as compared to BCR-ABL transduced cells alone. In addition, suppression of AHI-1 expression also resulted in a reduction in CFC output in lin−CD34+ stem/progenitor cells from CML patients (n=3). Strikingly, co-expression of Ahi-1 in BCR-ABL inducible BaF3 cells immediately reverses growth deficiencies exhibited by down-regulation of BCR-ABL in both semi-solid cultures (>50-fold) and in liquid suspension cultures in the absence of IL-3 (>30-fold), and is associated with sustained phosphorylation of BCR-ABL and enhanced activation of JAK2/STAT5. Moreover, a direct physical interaction between AHI-1 and BCR-ABL at endogenous levels has now been identified in CML cells; this interaction complex is also associated with a 120 kDa tyrosine phosphorylated protein. In addition, significant changes in imatinib (IM, an inhibitor of the BCR-ABL) response have been further demonstrated in Ahi-1 transduced BCR-ABL inducible BaF3 cells (P< 0.01) and in AHI-1 suppressed CML stem/progenitor cells (n=3). These findings provide strong evidence that Ahi-1/AHI-1 interacts with BCR-ABL to form an interaction complex and that this interaction complex further modulates altered BCR-ABL signaling and transforming activities of CML progenitor cells, including their response to IM.

Modulation of astrocyte P2Y1 receptors by the carboxyl terminal domain of the gap junction protein Cx43

Gap junction proteins, connexins, provide intercellular channels that allow ions and small signaling molecules to be transmitted to adjacent coupled cells. Besides this function, it is becoming apparent that connexins also exert channel-independent effects, which are likely mediated by processes involving protein-protein interactions. Although a number of connexin interacting proteins have been identified, only little is known about the functional consequences of such interactions. We have previously shown that deletion of the astrocytic gap junction protein, connexin43 (Cx43) causes a right-ward shift in the dose-response curve to P2Y1R agonists and decreased P2Y1R expression levels. To evaluate whether these changes were due to reduced gap junctional communication or to protein-protein interactions, Cx43-null astrocytes were transfected with full-length Cx43 and Cx43 domains, and P2Y1R function and expression levels evaluated. Results indicate that restoration of P2Y1R function is independent of gap junctional communication and that the Cx43 carboxyl terminus spanning the SH3 binding domain (260-280) participates in the rescue of P2Y1R pharmacological behavior (shifting to the left the P2Y1R dose-response curve) without affecting its expression levels. These results suggest that the Cx43 carboxyl-terminus domain provides a binding site for an intracellular molecule, most likely a member of the c-Src tyrosine kinase family, which affects P2Y1R-induced calcium mobilization. It is here proposed that a nonchannel function of Cx43 is to serve as a decoy for such kinases. Such modulation of P2Y1R is expected to influence several neural cell functions, especially under inflammation and neurodegenerative disorders where expression levels of Cx43 are decreased.

The Tyrosine Kinase Activity of c-Src Regulates Actin Dynamics and Organization of Podosomes in Osteoclasts

Podosomes are dynamic actin-rich structures composed of a dense F-actin core surrounded by a cloud of more diffuse F-actin. Src performs one or more unique functions in osteoclasts (OCLs), and podosome belts and bone resorption are impaired in the absence of Src. Using Src(-/-) OCLs, we investigated the specific functions of Src in the organization and dynamics of podosomes. We found that podosome number and the podosome-associated actin cloud were decreased in Src(-/-) OCLs. Videomicroscopy and fluorescence recovery after photobleaching analysis revealed that the life span of Src(-/-) podosomes was increased fourfold and that the rate of actin flux in the core was decreased by 40%. Thus, Src regulates the formation, structure, life span, and rate of actin polymerization in podosomes and in the actin cloud. Rescue of Src(-/-) OCLs with Src mutants showed that both the kinase activity and either the SH2 or the SH3 binding domain are required for Src to restore normal podosome organization and dynamics. Moreover, inhibition of Src family kinase activities in Src(-/-) OCLs by Src inhibitors or by expressing dominant-negative Src(K295M) induced the formation of abnormal podosomes. Thus, Src is an essential regulator of podosome structure, dynamics and organization.

COOH-terminal association of human smooth muscle calcium channel Cav1.2b with Src kinase protein binding domains: effect of nitrotyrosylation

The carboxyl terminus of the calcium channel plays an important role in the regulation of calcium entry, signal transduction, and gene expression. Potential protein-protein interaction sites within the COOH terminus of the L-type calcium channel include those for the SH3 and SH2 binding domains of c-Src kinase that regulates calcium currents in smooth muscle. In this study, we examined the binding sites involved in Src kinase-mediated phosphorylation of the human voltage-gated calcium channel (Ca(v)) 1.2b (hCav1.2b) and the effect of nitrotyrosylation. Cotransfection of human embryonic kidney (HEK)-293 cells with hCa(v)1.2b and c-Src resulted in tyrosine phosphorylation of the calcium channel, which was prevented by nitration of tyrosine residues by peroxynitrite. Whole cell calcium currents were reduced by 58 + 5% by the Src kinase inhibitor PP2 and 64 + 6% by peroxynitrite. Nitrotyrosylation prevented Src-mediated regulation of the currents. Glutathione S-transferase fusion protein of the distal COOH terminus of hCa(v)1.2b (1809-2138) bound to SH2 domain of Src following tyrosine phosphorylation, while binding to SH3 required the presence of the proline-rich motif. Site-directed mutation of Y(2134) prevented SH2 binding and resulted in reduced phosphorylation of hCa(v)1.2b. Within the distal COOH terminus, single, double, or triple mutations of Y(1837), Y(1861), and Y(2134) were constructed and expressed in HEK-293 cells. The inhibitory effects of PP2 and peroxynitrite on calcium currents were significantly reduced in the double mutant Y(1837-2134F). These data demonstrate that the COOH terminus of hCa(v)1.2b contains sites for the SH2 and SH3 binding of Src kinase. Nitrotyrosylation of these sites prevents Src kinase regulation and may be importantly involved in calcium influx regulation during inflammation.

SH3 Binding Motif 1 in Influenza A Virus NS1 Protein Is Essential for PI3K/Akt Signaling Pathway Activation

Recent studies have demonstrated that influenza A virus infection activates the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway by binding of influenza NS1 protein to the p85 regulatory subunit of PI3K. Our previous study proposed that two polyproline motifs in NS1 (amino acids 164 to 167 [PXXP], SH3 binding motif 1, and amino acids 213 to 216 [PPXXP], SH3 binding motif 2) may mediate binding to the p85 subunit of PI3K. Here we performed individual mutational analyses on these two motifs and demonstrated that SH3 binding motif 1 contributes to the interactions of NS1 with p85beta, whereas SH3 binding motif 2 is not required for this process. Mutant viruses carrying NS1 with mutations in SH3 binding motif 1 failed to interact with p85beta and induce the subsequent activation of PI3K/Akt pathway. Mutant virus bearing mutations in SH3 binding motif 2 exhibited similar phenotype as the wild-type (WT) virus. Furthermore, viruses with mutations in SH3 binding motif 1 induced more severe apoptosis than did the WT virus. Our data suggest that SH3 binding motif 1 in NS1 protein is required for NS1-p85beta interaction and PI3K/Akt activation. Activation of PI3K/Akt pathway is beneficial for virus replication by inhibiting virus induced apoptosis through phosphorylation of caspase-9.

Src kinase activity and SH2 domain regulate the dynamics of Src association with lipid and protein targets

Src functions depend on its association with the plasma membrane and with specific membrane-associated assemblies. Many aspects of these interactions are unclear. We investigated the functions of kinase, SH2, and SH3 domains in Src membrane interactions. We used FRAP beam-size analysis in live cells expressing a series of c-Src–GFP proteins with targeted mutations in specific domains together with biochemical experiments to determine whether the mutants can generate and bind to phosphotyrosyl proteins. Wild-type Src displays lipid-like membrane association, whereas constitutively active Src-Y527F interacts transiently with slower-diffusing membrane-associated proteins. These interactions require Src kinase activity and SH2 binding, but not SH3 binding. Furthermore, overexpression of paxillin, an Src substrate with a high cytoplasmic population, competes with membrane phosphotyrosyl protein targets for binding to activated Src. Our observations indicate that the interactions of Src with lipid and protein targets are dynamic and that the kinase and SH2 domain cooperate in the membrane targeting of Src.

In vivo tumorigenesis by Jaagsiekte sheep retrovirus (JSRV) requires Y590 in Env TM, but not full-length orfX open reading frame

Jaagsiekte retrovirus (JSRV) causes ovine pulmonary adenocarcinoma (OPA), a transmissible lung cancer of sheep. The envelope (Env) glycoprotein protein of JSRV functions as a dominant oncoprotein in vitro and in vivo. An SH2 binding domain (YXXM) in the cytoplasmic tail of the JSRV Env is one of the main determinants of viral transformation at least in vitro. In these studies, we report the first in vivo tests of site-specific mutants of JSRV in their natural host, the sheep. We show that, in vivo, JSRV 21 with the cytoplasmic tail YXXM mutated to DXXM did not cause disease nor detectable infection, indicating that this motif is absolutely required for virus replication and possibly transformation in vivo. In contrast, mutation of the JSRV open reading frame orfX, for which no function has yet been attributed, did not alter the disease induced by JSRV 21.

P53 codon 72 polymorphism in patients affected with ulcerative colitis

The p53 tumor suppressor protein plays a fundamental role in maintaining genomic integrity through its ability to arrest the cell cycle in G1 and induce apoptosis. The proapoptotic activity of p53 seems to be strictly related to proline-rich regions, homologous to the SH3 binding domain. In the literature, reported data suggest a role for polymorphism at codon 72 of p53 in the predisposition to neoplastic transformation, although the results are still controversial. In this study, we investigated Arg72Pro polymorphism of p53 and related this polymorphism to clinical parameters in patients affected with ulcerative colitis (UC). We studied 243 consecutive outpatients affected with well-established UC. The control group comprised 142 healthy blood donors, with age and sex comparable to those of the patients. p53 Pro/Pro was significantly related to the clinical course and duration of disease (odds ratio, 55.8 and 8.8, respectively). Nineteen of 24 patients with Pro homozygosity had a duration of disease >7 years. In contrast, 87 of 123 patients with Arg/Arg had short-standing UC (< or =7 yrs) and 66 of 96 with Arg/Pro had short-standing UC (chi-squared, 22.86; P < 0.0001). Thirty-four of 243 patients affected with UC had a positive family history for colorectal carcinoma (CRC). In those patients p53, Pro/Pro was significantly related to a family history of CRC (odds ratio, 38.1). These preliminary data suggest that polymorphism at codon 72 of the p53 gene influences the clinical course of UC, with continuous disease associated with p53 Pro homozygosity.