Biomedical subjects

K S Ravichandran

Publications and source records attributed to K S Ravichandran.

At least 19 recordsLinked to original sources

PubMed2001

CED-12/ELMO, a novel member of the CrkII/Dock180/Rac pathway, is required for phagocytosis and cell migration.

The C. elegans genes ced-2, ced-5, and ced-10, and their mammalian homologs crkII, dock180, and rac1, mediate cytoskeletal rearrangements during phagocytosis of apoptotic cells and cell motility. Here, we describe an additional member of this signaling pathway, ced-12, and its mammalian homologs, elmo1 and elmo2. In C. elegans, CED-12 is required for engulfment of dying cells and for cell migrations. In mammalian cells, ELMO1 functionally cooperates with CrkII and Dock180 to promote phagocytosis and cell shape changes. CED-12/ELMO-1 binds directly to CED-5/Dock180; this evolutionarily conserved complex stimulates a Rac-GEF, leading to Rac1 activation and cytoskeletal rearrangements. These studies identify CED-12/ELMO as an upstream regulator of Rac1 that affects engulfment and cell migration from C. elegans to mammals.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Signaling via Shc family adapter proteins.

The adapter protein Shc was initially identified as an SH2 containing proto-oncogene involved in growth factor signaling. Since then a number of studies in multiple systems have implicated a role for Shc in signaling via many different types of receptors, such as growth factor receptors, antigen receptors, cytokine receptors, G-protein coupled receptors, hormone receptors and integrins. In addition to the ubiquitous ShcA, two other shc gene products, ShcB and ShcC, which are predominantly expressed in neuronal cells, have also been identified. ShcA knockout mice are embryonic lethal and have clearly suggested an important role for ShcA in vivo. Based on dominant negative studies and mouse embryos deficient in ShcA, a clear role for Shc in leading to mitogen activated protein kinase (MAPK) activation has been established. However MAPK activation may not be the sole function of Shc proteins. Although Shc has also been linked to other signaling events such as c-Myc activation and cell survival, the mechanistic understanding of these signaling events remains poorly characterized. Given the apparently central role that Shc plays signaling via many receptors, delineating the precise mechanism(s) of Shc-mediated signaling may be critical to our understanding of the effects mediated through these receptors.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Evidence for a conserved role for CRKII and Rac in engulfment of apoptotic cells.

Apoptosis or programmed cell death occurs in multicellular organisms throughout life. The removal of apoptotic cells by phagocytes prevents secondary necrosis and inflammation and also plays a key role in tissue remodeling and regulating immune responses. The molecular mechanisms that regulate the engulfment of apoptotic cells are just beginning to be elucidated. Recent genetic studies in the nematode Caenorhabditis elegans have implicated at least six genes in the removal of apoptotic cell corpses. The gene products of ced-2, ced-5, and ced-10 are thought to be part of a pathway that regulates the reorganization of the cytoskeleton during engulfment. The adapter proteins CrkII and Dock180 and the small GTPase Rac represent the mammalian orthologues of the ced-2, ced-5 and ced-10 gene products, respectively. It is not known whether CrkII, Dock180, or Rac proteins have any role during engulfment in mammalian cells. Here we show, using stable cell lines and transient transfections, that overexpression of wild-type CrkII or an activated form of Rac1 enhances engulfment. Mutants of CrkII failed to mediate this increased engulfment. The higher CrkII-mediated uptake was inhibited by coexpression of a dominant negative form of Rac1 but not by a dominant a negative Rho protein; this suggested that Rac functions downstream of CrkII in this process, which is consistent with genetic studies in the worm that place ced-10 (rac) downstream of ced-2 (crk) in cell corpse removal. Taken together, these data suggest that CED-2/CrkII and CED-10/Rac are part of an evolutionarily conserved pathway in engulfment of apoptotic cells.

Animals↗

PubMed2001

Design and use of an inducibly activated human immunodeficiency virus type 1 Nef to study immune modulation.

The Nef protein of the human immunodeficiency virus type 1 (HIV-1) has been shown to enhance the infectivity of virus particles, downmodulate cell surface proteins, and associate with many intracellular proteins that are thought to facilitate HIV infection. One of the challenges in defining the molecular events regulated by Nef has been obtaining good expression of Nef protein in T cells. This has been attributed to effects of Nef on cell proliferation and apoptosis. We have designed a Nef protein that is readily expressed in T-cell lines and whose function is inducibly activated. It is composed of a fusion between full-length Nef and the estrogen receptor hormone-binding domain (Nef-ER). The Nef-ER is kept in an inactive state due to steric hindrance, and addition of the membrane-permeable drug 4-hydroxytamoxifen (4-HT), which binds to the ER domain, leads to inducible activation of Nef-ER within cells. We demonstrate that Nef-ER inducibly associates with the 62-kDa Ser/Thr kinase and is localized to specific membrane microdomains (lipid rafts) only after activation. Using this inducible Nef, we also compared the specific requirements for CD4 and HLA-A2 downmodulation in a SupT1 T-cell line. Half-maximal downmodulation of cell surface CD4 required very little active Nef-ER and occurred as early as 4 h after addition of 4-HT. In contrast, 50% downmodulation of HLA-A2 by Nef required 16 to 24 h and about 50- to 100-fold-greater concentrations of 4-HT. These data suggest that HLA-A2 downmodulation may require certain threshold levels of active Nef. The differential timing of CD4 and HLA-A2 downmodulation may have implications for HIV pathogenesis and immune evasion.

Animals↗

PubMed2000

p135 src homology 2 domain-containing inositol 5'-phosphatase (SHIPbeta ) isoform can substitute for p145 SHIP in fcgamma RIIB1-mediated inhibitory signaling in B cells.

The inositol 5'-phosphatase, SHIP (also referred to as SHIP-1 or SHIPalpha), is expressed in all cells of the hematopoietic lineage. Depending on the cell type being investigated and the state of differentiation, SHIP isoforms of several different molecular masses (170, 160, 145, 135, 125, and 110 kDa) have been seen in immunoblots. However, the function of the individual isoforms and the effect of expressing multiple isoforms simultaneously are not understood. Some of these SHIP isoforms have recently been characterized at the level of primary sequence. In this report, we investigated the function of the recently characterized 135-kDa SHIP isoform (SHIPbeta), which appears to possess the catalytic domain but lacks some of the protein-protein interaction motifs at the C terminus. By reconstituting SHIP-deficient DT40 B cells with either SHIPbeta or the better-characterized p145 SHIPalpha, we addressed the function of SHIPbeta in the complete absence of SHIPalpha. We observed that SHIPbeta had enzymatic activity comparable with SHIPalpha and that SHIPbeta was able to reconstitute F(c)gammaRIIB1-mediated inhibition of B cell receptor-induced signaling events such as calcium flux and Akt and mitogen-activated protein kinase activation. SHIPbeta was readily phosphorylated in response to B cell receptor cross-linking with the inhibitory receptor F(c)gammaRIIB1 and SHIPbeta also interacted with the adapter protein Shc. During these studies we also observed that the SHIPalpha or SHIPbeta interaction with Grb2 is not required for F(c)gammaRIIB1-mediated inhibition of calcium flux. These data suggest that SHIPbeta, which is normally expressed in B cells along with SHIPalpha, functions comparably with SHIPalpha and that these two isoforms are not likely to be antagonistic in their function in vivo.

Animals↗

PubMed2000

SHIP inhibits Akt activation in B cells through regulation of Akt membrane localization.

Activation of the serine/threonine kinase Akt and the regulation of its activation are recognized as critical in controlling proliferative/survival signals via many hematopoietic receptors. In B lymphocytes, the B-cell receptor (BCR)-mediated activation of Akt is attenuated by co-cross-linking of BCR with the inhibitory receptor Fc gamma RIIB1, and the binding of the SH2 domain-containing inositol phosphatase, SHIP, to Fc gamma RIIB1. Because SHIP dephosphorylates phosphatidylinositol 3,4,5-trisphosphate (PIP3) and activation of Akt requires PIP3, the destruction of this phospholipid has been proposed as the mechanism for Akt inhibition. However, upstream kinases that activate Akt, such as PDK1, also require PIP3 for activation. In this report, we addressed whether SHIP inhibits Akt directly at the level of Akt recruitment to the membrane, indirectly through PDK recruitment/phosphorylation of Akt, or both. We generated stable B-cell lines expressing a regulatable, but constitutively membrane-bound Akt that still required PDK-dependent phosphorylation for activation. Several lines of evidence suggested that activation of this membrane-targeted Akt is not inhibited by Fc gamma RIIB1/SHIP and that PDK is not a target for SHIP-mediated inhibition. These data demonstrate that SHIP inhibits Akt primarily through regulation of Akt membrane localization. We also observed during these studies that Fc gamma RIIB1/SHIP does not inhibit p70(S6k) activation, even though several other PIP3-dependent events were down-regulated. Because the enhanced activation of Akt in the absence of SHIP correlates with hyperproliferation in the myeloid lineage, our data have implications for SHIP and Akt-dependent regulation of proliferation in the hematopoietic lineage. (Blood. 2000;96:1449-1456)

Animals↗

PubMed2000

Urokinase-type plasminogen activator stimulates the Ras/Extracellular signal-regulated kinase (ERK) signaling pathway and MCF-7 cell migration by a mechanism that requires focal adhesion kinase, Src, and Shc. Rapid dissociation of GRB2/Sps-Shc complex is associated with the transient phosphorylation of ERK in urokinase-treated cells.

Urokinase-type plasminogen activator (uPA) stimulates MCF-7 cell migration by binding to the UPA receptor and activating the Ras-extracellular signal-regulated kinase (Ras-ERK) signaling pathway. Studies presented here show that soluble uPA receptor and a peptide derived from the linker region between domains 1 and 2 of the uPA receptor also stimulate cellular migration via a mitogen-activated protein kinase/ERK kinase (MEK)-dependent pathway. Signaling proteins that function upstream of Ras in uPA- stimulated cells remain undefined. To address this problem, we transfected MCF-7 cells to express the noncatalytic carboxylterminal domain of focal adhesion kinase (FAK), FAK(Y397F), kinase-defective c-Src, or Shc FFF, all of which express dominant-negative activity. In each case, ERK phosphorylation and cellular migration in response to uPA were blocked. Both activities were rescued by co-transfecting the cells to express constitutively active MEK1, indicating that FAK, c-Src, and Shc are upstream of MEK. Shc was tyrosine-phosphorylated in uPA-treated cells. The level of phosphorylated Shc was increased within 1 min and remained increased for at least 30 min. Sos co-immunoprecipitated with Shc in cells that were treated with uPA for 1-2.5 min, probably reflecting the formation of Shc-Grb2/Sos complex; however, by 10 min, co-immunoprecipitation of Sos with Shc was no longer observed. Rapid dissociation of Sos from Shc represents a possible mechanism for the transient phosphorylation of ERK in uPA-treated MCF-7 cells.

Adaptor Proteins, Signal Transducing↗

PubMed2000

A requirement for lipid rafts in B cell receptor induced Ca(2+) flux.

Although the major biochemical events triggered by ligation of the B-cell receptor (BCR) have been well defined [1] [2], little is known about the spatio-temporal organization of BCR signaling components within the cell membrane and the mechanisms by which signaling specificity is achieved. Partitioning of signaling complexes into specialized domains in the plasma membrane may provide a mechanism for channeling specific stimuli into distinct signaling pathways. Here, we report that multiple tyrosine-phosphorylated proteins accumulate transiently upon BCR activation in detergent-insoluble membrane microdomains known as lipid rafts. We found an activation-dependent translocation to the rafts of the BCR itself, as well as phospholipase Cgamma2 (PLCgamma2), an enzyme critical for BCR-induced Ca(2+) flux in B cells. An intact raft structure was required for BCR-induced tyrosine phosphorylation of PLCgamma2 and the induction of Ca(2+) flux. Taken together, these data provide a functional role for lipid rafts in BCR signaling.

B-Lymphocytes↗

PubMed2000

Identification and characterization of a dimerization domain in CED-6, an adapter protein involved in engulfment of apoptotic cells.

Phagocytosis of apoptotic cells is a key step in the completion of programmed cell death that occurs throughout life in multicellular organisms. The molecular events involved in clearance of apoptotic cells are just beginning to be elucidated. Recently, CED-6, an adapter protein involved in engulfment has been cloned in Caenorhabditis elegans and in humans. CED-6 is composed of a phosphotyrosine-binding (PTB) domain and a proline-rich C-terminal domain with no apparent catalytic domain. Since PTB domains, originally identified in Shc, mediate intracellular signaling downstream of cell surface receptors, CED-6 has also been proposed to mediate intracellular signals leading to engulfment. In this report, we demonstrate that CED-6 dimerizes through a leucine zipper domain that is immediately adjacent to the PTB domain. Several lines of evidence based on co-immunoprecipitation studies, yeast two-hybrid assays, and gel filtration studies suggest that CED-6 exists as a dimer in vivo. Through mutational analyses, we show that the leucine zipper is necessary and sufficient for CED-6 dimerization and that this dimerization is conserved among C. elegans, rodent, and human CED-6 proteins. We propose that dimerization may have unique implications for ligand binding via CED-6 and its function during the phagocytosis of apoptotic cells.

Amino Acid Sequence↗

PubMed2000

Essential role for the C-terminal noncatalytic region of SHIP in FcgammaRIIB1-mediated inhibitory signaling.

The inositol phosphatase SHIP binds to the FcgammaRIIB1 receptor and plays a critical role in FcgammaRIIB1-mediated inhibition of B-cell proliferation and immunoglobulin synthesis. The molecular details of SHIP function are not fully understood. While point mutations of the signature motifs in the inositol phosphatase domain abolish SHIP's ability to inhibit calcium flux in B cells, little is known about the function of the evolutionarily conserved, putative noncatalytic regions of SHIP in vivo. In this study, through a systematic mutagenesis approach, we identified the inositol phosphatase domain of SHIP between amino acids 400 and 866. Through reconstitution of a SHIP-deficient B-cell line with wild-type and mutant forms of SHIP, we demonstrate that the catalytic domain alone is not sufficient to mediate FcgammaRIIB1/SHIP-dependent inhibition of B-cell receptor signaling. Expression of a truncation mutant of SHIP that has intact phosphatase activity but lacks the last 190 amino acids showed that the noncatalytic region in the C terminus is essential for inhibitory signaling. Mutation of two tyrosines within this C-terminal region, previously identified as important in binding to Shc, showed a reduced inhibition of calcium flux. However, studies with an Shc-deficient B-cell line indicated that Shc-SHIP complex formation is not required and that other proteins that bind these tyrosines may be important in FcgammaRIIB1/SHIP-mediated calcium inhibition. Interestingly, membrane targeting of SHIP lacking the C terminus is able to restore this inhibition, suggesting a role for the C terminus in localization or stabilization of SHIP interaction at the membrane. Taken together, these data suggest that the noncatalytic carboxyl-terminal 190 amino acids of SHIP play a critical role in SHIP function in B cells and may play a similar role in several other receptor systems where SHIP functions as a negative regulator.

Antigens, CD↗

PubMed1999

Requirement for Shc in TCR-mediated activation of a T cell hybridoma.

Engagement of the TCR determines the fate of T cells to activate their functional programs, proliferate, or undergo apoptosis. The intracellular signal transduction pathways that dictate the specific outcome of receptor engagement have only been partially elucidated. The adapter protein, Shc, is involved in cytokine production, mitogenesis, transformation, and apoptosis in different cell systems. We found that Shc becomes phosphorylated on tyrosine residues upon stimulation of the TCR in DO11.10 hybridoma T cells; therefore, we investigated the role of Shc in activation-induced cell death in these cells by creating a series of stably transfected cell lines. Expression of Shc-SH2 (the SH2 domain of Shc) or Shc-Y239/240F (full-length Shc in which tyrosines 239 and 240 have been mutated to phenylalanine) resulted in the inhibition of activation-induced cell death and Fas ligand up-regulation after TCR cross-linking. Expression of wild-type Shc or Shc-Y317F had no significant effect. In addition, we found that Shc-SH2 and Shc-Y239/240F, but not Shc-Y317F, inhibited phosphorylation of extracellular signal-regulated protein kinase and production of IL-2 after TCR cross-linking. These results indicate an important role for Shc in the early signaling events that lead to activation-induced cell death and IL-2 production after TCR activation.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Phosphatidylinositol 3-kinase-dependent and -independent cytolytic effector functions.

Two distinct forms of short-term cytolysis have been described for CD8+ CTLs, the perforin/granzyme- and Fas ligand/Fas (CD95 ligand (CD95L)/CD95)-mediated pathways. However, the difference in signal transduction events leading to these cytolytic mechanisms remains unclear. We used wortmannin, an irreversible antagonist of phosphatidylinositol 3-kinase (PI3-K) activity, to investigate the role of PI3-K in influenza-specific CD8+ CTL cytolytic effector function. We found that the addition of wortmannin at concentrations as low as 1 nM significantly inhibited both the Ag/MHC-induced cytolysis of CD95- target cells and serine esterase release. In strong contrast, W did not inhibit the Ag/MHC-induced CD95L expression or the CD95L/CD95-mediated cytolysis of CD95+ targets. A combination of wortmannin and blocking mAb against CD95L inhibited the cytolysis of CD95+ targets, indicating that the wortmannin-independent cytolysis was due to CD95L/CD95 mediated cytolysis. These findings suggest a differential role for PI3-K in mediating cytolysis and, thus far, the earliest difference between perforin/granzyme- and CD95L/CD95-dependent cytolysis. Our data reinforce the idea of a TCR with modular signal transduction pathways that can be triggered or inhibited selectively, resulting in differential effector function.

Androstadienes↗

PubMed1999

Neuregulin-increased expression of acetylcholine receptor epsilon-subunit gene requires ErbB interaction with Shc.

Selective transcription of acetylcholine receptor (AChR) subunit genes by neuregulin is one of the mechanisms involved in the synaptic localization of AChRs to the neuromuscular junction. Neuregulin stimulates ErbB receptor tyrosine kinases and subsequently activates the Ras/ERK pathway, which is required for neuregulin-mediated induction of AChR subunit genes in muscle cells and synapse-specific expression in vivo. Here we investigated the neuregulin transduction mechanism that leads to ERK activation after ErbB receptor tyrosine phosphorylation. Neuregulin increases the association of the adaptor proteins Grb2 and Shc with both ErbB2 and ErbB3 in C2C12 muscle cells. Dephosphorylation of the tyrosine-phosphorylated ErbB proteins abolished their association with both Grb2 and Shc, suggesting a tyrosine phosphorylation-dependent interaction. The interaction of Shc with the ErbB receptors is mediated by Shc's phosphotyrosine-binding domain. In addition, neuregulin increased tyrosine phosphorylation of Shc. Mutagenesis approaches demonstrated that tyrosine phosphorylation of Shc is required for neuregulin induction of AChR subunit gene expression. Taken together, these data indicate that the interaction of ErbB receptors with Grb2 alone is insufficient for neuregulin-activated transcription, but that ErbB receptor signaling via Shc is necessary and important.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Shc dominant negative disrupts cell cycle progression in both G0-G1 and G2-M of ErbB2-positive breast cancer cells.

The Shc protein helps to transmit signals from receptor and cytoplasmic tyrosine kinases to Ras. We have shown that several breast cancer cell lines (MDA-MB-453, BT474, MDA-MB-361, and SKBR3), which overexpress the ErbB2 receptor tyrosine kinase, contain constitutively tyrosine phosphorylated Shc. To investigate the role of Shc in these cells, we transfected them with a Shc-Y317F dominant-negative mutant defective in signaling to Ras. The transfectants were unable to form stable colonies, suggesting a critical role for Shc in the proliferation of these cells. In contrast, dominant-negative Shc transfectants of the nontransformed breast epithelial cell line HBL-100 grew normally. Surprisingly, cell cycle analysis of transfected SKBR3 cells suggested that the cells were blocked not only in G0-G1, but also in G2-M. The G2-M block was unexpected because Shc-Y317 is downstream of receptor tyrosine kinases that drive the early events in the cell cycle. Both the G0-G1 and G2-M arrest were rescued by transfection with wild-type Shc or oncogenic Ras 12V. Rescue by Ras suggests that Shc Y317 signals upstream of Ras, and that Shc to Ras effector pathways are involved in G2-M, although confirmation awaits a detailed molecular analysis. Most importantly, this work provides the first evidence for Shc involvement in G2-M.

Breast↗

PubMed1998

The inositol phosphatase SHIP inhibits Akt/PKB activation in B cells.

The serine-threonine kinase Akt/PKB is activated downstream of phosphatidylinositol 3-kinase in response to several growth factor stimuli and has been implicated in the promotion of cell survival. Although both phosphatidylinositol 3,4,5-trisphosphate (PIP3) and phosphatidylinositol 3,4-bisphosphate (PI 3,4-P2) have been implicated in the regulation of Akt activity in vitro, the relative roles of these two phospholipids in vivo are not well understood. Co-ligation of the B cell receptor (BCR) and the inhibitory FcgammaRIIB1 on B cells results in the recruitment of the 5'-inositol phosphatase SHIP to the signaling complex. Since SHIP is known to cleave PIP3 to generate PI 3,4-P2 both in vivo and in vitro, and Akt activity has been reported to be regulated by either PIP3 or PI 3,4-P2, we hypothesized that recruitment of SHIP through FcgammaRIIB1 co-cross-linking to the BCR in B cells might regulate Akt activity. The nature of this regulation, positive or negative, might also reveal the relative contribution of PIP3 and PI 3,4-P2 to Akt activation in vivo. Here we report that Akt is activated by stimulation through the BCR in a phosphatidylinositol 3-kinase-dependent manner and that this activation is inhibited by co-cross-linking of the BCR to FcgammaRIIB1. Using mutants of FcgammaRIIB1 and SHIP-deficient B cells, we demonstrate that inhibition of Akt activity is mediated by the immune cell tyrosine-based inhibitory motif within FcgammaRIIB1 as well as SHIP. The SHIP-dependent inhibition of Akt activation also suggests that PIP3 plays a greater role in Akt activation than PI 3,4-P2 in vivo.

Animals↗

PubMed1998

Roles of Lck, Syk and ZAP-70 tyrosine kinases in TCR-mediated phosphorylation of the adapter protein Shc.

The adapter protein Shc has been implicated in mitogenic signaling via growth factor receptors, antigen receptors and cytokine receptors. Recent studies have suggested that tyrosine phosphorylation of Shc may play a key role in T lymphocyte proliferation via interaction of phosphorylated Shc with downstream molecules involved in activation of Ras and Myc proteins. However, the sites on Shc that are tyrosine phosphorylated in response to TCR engagement and the ability of different T cell tyrosine kinases to phosphorylate Shc have not been defined. In this report, we show that during TCR signaling, the tyrosines Y239, Y240 and Y317 of Shc are the primary sites of tyrosine phosphorylation. Mutation of all three tyrosines completely abolished tyrosine phosphorylation of Shc following TCR stimulation. Our data also suggest that multiple T cell tyrosine kinases contribute to tyrosine phosphorylation on Shc. In T cells, CD4/Lck-dependent tyrosine phosphorylation on Shc was markedly diminished when Y317 was mutated, suggesting a preference of Lck for the Y317 site. The syk-family kinases (Syk and ZAP-70) were able to phosphorylate the Y239 and Y240 sites, and less efficiently the Y317 site. Moreover, co-expression of Syk or ZAP-70 with Lck resulted in enhanced phosphorylation of Shc on all three sites, suggesting a synergy between the syk-family and scr-family kinases. Of the two potential Grb2 binding sites (Y239 and Y317), Y239 appears to play a greater role in recruiting Sos through Grb2. These studies have implications for Ras activation and mitogenic signaling during T cell activation.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Epidermal growth factor receptor and the adaptor protein p52Shc are specific substrates of T-cell protein tyrosine phosphatase.

T-cell protein tyrosine phosphatase (TCPTP) exists as two forms generated by alternative splicing: a 48-kDa endoplasmic reticulum (ER)-associated form (TC48) and a 45-kDa nuclear form (TC45). To identify TCPTP substrates, we have generated substrate-trapping mutants, in which the invariant catalytic acid of TCPTP (D182) is mutated to alanine. The TCPTP D182A substrate-trapping mutants were transiently overexpressed in COS cells, and their ability to form complexes with tyrosine-phosphorylated (pTyr) proteins was assessed. No pTyr proteins formed complexes with wild-type TCPTP. In contrast, TC48-D182A formed a complex in the ER with pTyr epidermal growth factor receptor (EGFR). In response to EGF, TC45-D182A exited the nucleus and accumulated in the cytoplasm, where it bound pTyr proteins of approximately 50, 57, 64, and 180 kDa. Complex formation was disrupted by vanadate, highlighting the importance of the PTP active site in the interaction and supporting the characterization of these proteins as substrates. Of these TC45 substrates, the approximately 57- and 180-kDa proteins were identified as p52Shc and EGFR, respectively. We examined the effects of TC45 on EGFR signaling and observed that it did not modulate EGF-induced activation of p42Erk2. However, TC45 inhibited the EGF-induced association of p52Shc with Grb2, which was attributed to the ability of the PTP to recognize specifically p52Shc phosphorylated on Y239. These results indicate that TC45 recognizes not only selected substrates in a cellular context but also specific sites within substrates and thus may regulate discrete signaling events.

3T3 Cells↗

PubMed1997

A comparative analysis of the phosphoinositide binding specificity of pleckstrin homology domains.

Pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains are structurally related regulatory modules that are present in a variety of proteins involved in signal transduction, such as kinases, phospholipases, GTP exchange proteins, and adapter proteins. Initially these domains were shown to mediate protein-protein interactions, but more recently they were also found to bind phosphoinositides. Most studies to date have focused on binding of PH domains to phosphatidylinositol (PtdIns)-4-P and PtdIns-4,5-P2 and have not considered the lipid products of phosphoinositide 3-kinase: PtdIns-3-P, PtdIns-3,4-P2, and PtdIns-3,4,5-P3. Here we have compared the phosphoinositide specificity of six different PH domains and the Shc PTB domain using all five phosphoinositides. We show that the Bruton's tyrosine kinase PH domain binds to PtdIns-3,4, 5-P3 with higher affinity than to PtdIns-4,5-P2, PtdIns-3,4-P2 or inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4). This selectivity is decreased by the xid mutation (R28C). Selective binding of PtdIns-3,4,5-P3 over PtdIns-4,5-P2 or PtdIns-3,4-P2 was also observed for the amino-terminal PH domain of T lymphoma invasion and metastasis protein (Tiam-1), the PH domains of Son-of-sevenless (Sos) and, to a lesser extent, the PH domain of the beta-adrenergic receptor kinase. The oxysterol binding protein and beta-spectrin PH domains bound PtdIns-3,4,5-P3 and PtdIns-4,5-P2 with similar affinities. PtdIns-3,4,5-P3 and PtdIns-4,5-P2 also bound to the PTB domain of Shc with similar affinities and lipid binding was competed with phosphotyrosine (Tyr(P)-containing peptides. These results indicate that distinct PH domains select for different phosphoinositides.

Agammaglobulinaemia Tyrosine Kinase↗