Recruitment and phosphorylation of SH2-containing inositol phosphatase and Shc to the B-cell Fc gamma immunoreceptor tyrosine-based inhibition motif peptide motif

Recently, we and others have demonstrated that negative signaling in B cells selectively induces the tyrosine phosphorylation of a novel inositol polyphosphate phosphatase, p145SHIP. In this study, we present data indicating that p145SHIP binds directly a phosphorylated motif, immunoreceptor tyrosine-based inhibition motif (ITIM), present in the cytoplasmic domain of Fc gammaRIIB1. Using recombinant SH2 domains, we show that binding is mediated via the Src homology region 2 (SH2)-containing inositol phosphatase (SHIP) SH2 domain. SHIP also bound to a phosphopeptide derived from CD22, raising the possibility that SHIP contributes to negative signaling by this receptor as well as Fc gammaRIIB1. The association of SHIP with the ITIM phosphopeptide was activation independent, while coassociation with Shc was activation dependent. Furthermore, experiments with Fc gammaRIIB1-deficient B cells demonstrated a genetic requirement for expression of Fc gammaRIIB1 in the induction of SHIP phosphorylation and its interaction with Shc. Based on these results, we propose a model of negative signaling in which co-cross-linking of surface immunoglobulin and Fc gammaRIIB1 results in sequential tyrosine phosphorylation of the ITIM, recruitment and phosphorylation of p145SHIP, and subsequent binding of Shc.     

The inositol 5'-phosphatase SHIP binds to immunoreceptor signaling motifs and responds to high affinity IgE receptor aggregation

Immunoreceptors such as the high affinity IgE receptor, FcepsilonRI, and T-cell receptor-associated proteins share a common motif, the immunoreceptor tyrosine-based activation motif (ITAM). We used the yeast tribrid system to identify downstream effectors of the phosphorylated FcepsilonRI ITAM-containing subunits beta and gamma. One novel cDNA was isolated that encodes a protein that is phosphorylated on tyrosine, contains a Src-homology 2 (SH2) domain, inositolpolyphosphate 5-phosphatase activity, three NXXY motifs, several proline-rich regions, and is called SHIP. Mutation of the conserved tyrosine or leucine residues within the FcepsilonRI beta or gamma ITAMs eliminates SHIP binding and indicates that the SHIP-ITAM interaction is specific. SHIP also binds to ITAMs from the CD3 complex and T cell receptor zeta chain in vitro. SHIP protein possesses both phosphatidylinositol-3,4,5-trisphosphate 5'-phosphatase and inositol-1,3,4,5-tetrakisphosphate 5'-phosphatase activity. Phosphorylation of SHIP by a protein-tyrosine kinase, Lck, results in a reduction in enzyme activity. FcepsilonRI activation induces the association of several tyrosine phosphoproteins with SHIP. SHIP is constitutively tyrosine-phosphorylated and associated with Shc and Grb2. These data suggest that SHIP may serve as a multifunctional linker protein in receptor activation.     

Thrombopoietin induces association of Crkl with STAT5 but not STAT3 in human platelets

Crkl, a 39-kD SH2, SH3 domain-containing adapter protein, is constitutively tyrosine phosphorylated in hematopoietic cells from chronic myelogenous leukemia (CML) patients. We recently reported that thrombopoietin induces tyrosine phosphorylation of Crkl in normal platelets. In this study, we demonstrate that thrombopoietin induces association of Crkl with a tyrosine phosphorylated 95- to 100-kD protein in platelets and in UT7/TPO cells, a thrombopoietin-dependent megakaryocytic cell line. With specific antibodies against STAT5, we demonstrate that the 95- to 100-kD protein in Crkl immunoprecipitates is STAT5. This coimmunoprecipitation was specific in that Crkl immunoprecipitates do not contain STAT3, although STAT3 becomes tyrosine phosphorylated in thrombopoietin-stimulated platelets. The coimmunoprecipitaion of Crkl with STAT5 was inhibited by the immunizing peptide for Crkl antisera or phenyl phosphate (20 mmol/L). After denaturing of Crkl immunoprecipitates, Crkl was still immunoprecipitated by Crkl antisera. However, coimmunoprecipitation of STAT5 was not observed. Coincident with STAT5 tyrosine phosphorylation, thrombopoietin induces activation of STAT5 DNA-binding activity as demonstrated by electrophoretic mobility shift assays (EMSA). Using a beta-casein promoter STAT5 binding site as a probe, we have also demonstrated that Crkl antisera supershift the STAT5-DNA complex, suggesting that Crkl is a component of the complex in the nucleus. Furthermore, interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin also induce Crkl-STAT5 complex formation in responding cells in a stimulation-dependent manner. In vitro, glutathione S-transferase (GST)-Crkl bound to STAT5 inducibly through its SH2 domain. These results indicate that thrombopoietin, IL-3, GM-CSF, and erythropoietin commonly induce association of STAT5 and Crkl and that the complex translocates to the nucleus and binds to DNA. Interestingly, such association between STAT5 and Crkl was not observed in cytokine-stimulated murine cells, suggesting an intriguing possibility that components of the human STAT5-DNA complex may be different from those of the murine counterpart.     

Shc interaction with Src homology 2 domain containing inositol phosphatase (SHIP) in vivo requires the Shc-phosphotyrosine binding domain and two specific phosphotyrosines on SHIP

The adapter protein Shc has been implicated in mitogenic signaling via growth factor receptors, cytokine receptors, and antigen receptors on lymphocytes. Besides the well characterized interaction of Shc with molecules involved in Ras activation, Shc also associates with a 145-kDa tyrosine-phosphorylated protein upon triggering via antigen receptors and many cytokine receptors. This 145-kDa protein has been recently identified as an SH2 domain containing 5'-inositol phosphatase (SHIP) and has been implicated in the regulation of growth and differentiation in hematopoietic cells. In this report, we have addressed the molecular details of the interaction between Shc and SHIP in vivo. During T cell receptor signaling, tyrosine phosphorylation of SHIP and its association with Shc occurred only upon activation. We demonstrate that the phosphotyrosine binding domain of Shc is necessary and sufficient for its association with tyrosine-phosphorylated SHIP. Through site-directed mutagenesis, we have identified two tyrosines on SHIP, Tyr-917, and Tyr-1020, as the principal contact sites for the Shc-phosphotyrosine binding domain. Our data also suggest a role for the tyrosine kinase Lck in phosphorylation of SHIP. We also show that the SH2 domain of SHIP is dispensable for the Shc-SHIP interaction in vivo. These data have implications for the localization of the Shc.SHIP complex and regulation of SHIP function during T cell receptor signaling.     

p85 subunit of PI3 kinase does not bind to human Flt3 receptor, but associates with SHP2, SHIP, and a tyrosine-phosphorylated 100-kDa protein in Flt3 ligand-stimulated hematopoietic cells

Flt3/Flk2 belongs to class III receptor tyrosine kinases. Like other members of type III receptor tyrosine kinases, murine Flt3 induces tyrosine phosphorylation of p85 and subsequently activation of PI3 kinase upon FL binding. While p85 binds murine Flt3 at Y958 in the carboxyl terminus of the receptor, human Flt3 does not have a potential p85-binding site in the carboxyl terminus. In this study, we examined whether p85 binds to human Flt3 in Baf3/Flt3 and THP-1 cells. In contrast to murine Flt3, p85 is not tyrosine phosphorylated after FL stimulation, nor does it bind Flt3 in both cell lines. Instead p85 associates inducibly with tyrosine phosphorylated SHP-2 and constitutively with SHIP and two tyrosine phosphorylated proteins with molecular weights about 100-kDa (p100) and 120-kDa (p120) in Baf3/Flt3 cells. The p100 associates with both p85 and SHP-2. In THP-1 cells, p85 associates inducibly with tyrosine phosphorylated SHIP, p100 and p120. These results indicate that p85 does not bind human Flt3, but forms a complex with SHP-2, SHIP, p100 and p120 in hematopoietic cells.     

Tyrosine phosphorylation and association with phospholipase C gamma-1 of the GAP-associated 62-kD protein after CD2 stimulation of Jurkat T cell

Numerous substrates are tyrosine phosphorylated upon CD2 stimulation of human Jurkat T cells using a mitogenic pair of CD2 monoclonal antibodies, including the phospholipase C (PLC)gamma-1-p35/36 complex. Most of these substrates are identically tyrosine phosphorylated after CD3 ligation, suggesting that both stimuli share the same biochemical pathway. We show, however, in this report that a 63-kD protein is specifically phosphorylated on tyrosine residues after ligation of the CD2 molecule. The tyrosine phosphorylation of p63 can be induced independently of other substrates when using a single CD2 mAb recognizing the D66 epitope of the molecule. Importantly, this CD2-induced tyrosine phosphorylation of p63 can also occur in the absence of the CD3 zeta chain membrane expression, and is also distinct from the protein tyrosine kinases p56lck and p59fyn. We demonstrate, moreover, that p63 is physically linked with PLC gamma-1 and p35/36 upon CD2 stimulation. Finally, we also show that a 62-kD protein coimmunoprecipitating with the p21ras GTPase activating protein (GAP) is heavily tyrosine phosphorylated only after CD2 stimulation. This ultimately suggests that p63 may represent in fact the 62-kD protein that associates with GAP after tyrosine phosphorylation. Taken together, these results demonstrate the occurrence in Jurkat cells of a tyrosine kinase pathway specifically coupled to the CD2 molecule. They also suggest a function of the p62-GAP-associated protein as a link between PLC gamma-1 and p21ras activation pathways after CD2 activation.     

Meta-synthesis of qualitative analyses of caring: defining a therapeutic model of nursing

SHIP is a SH2 domain-containing inositol polyphosphatase that is selectively tyrosine phosphorylated and associated with the adapter protein Shc in B lymphocytes upon co-crosslinking surface immunoglobulin and Fc gamma RIIB1. We previously observed that this stimulation condition is associated with a reduction in the interaction of Grb2 with phosphorylated Shc, an enhanced interaction of Shc with SHIP, and a block in the Ras signaling pathway. We proposed that the SH2 domain of SHIP competes with Grb2 in binding to phospho-Shc, resulting in a block in Ras signaling. To test this model, we examined the mode of SHIP-Shc interaction. Using recombinant Shc and SHIP interaction domains and purified Shc and SHIP phosphopeptides, we show that the interaction is bi-dentate such that the SH2 domain of SHIP recognizes phosphorylated Y317 and doubly-phosphorylated Y239/Y240 of Shc and the Shc PTB domain recognizes phosphorylated NPxpY motifs within SHIP. We observed no role for the Shc SH2 domain in the interaction. These findings are consistent with our earlier model that SHIP and Grb2 compete for binding to phospho-Shc and support the notion that, in addition to the hydrolysis of inositol phosphates and phospholipids, SHIP contributes to anti-proliferative biochemistry by blocking protein-protein interactions.     

BMX, a novel nonreceptor tyrosine kinase gene of the BTK/ITK/TEC/TXK family located in chromosome Xp22.2

The Bmx sequence was identified and cloned during our search for novel tyrosine kinase genes expressed in human bone marrow cells. Bmx cDNA comprises a long open reading frame of 675 amino acids, containing one SH3, one SH2 and one tyrosine kinase domain, which are about 70% identical with Btk, Itk and Tec and somewhat less with Txk tyrosine kinase sequences. The amino terminal sequences of these four tyrosine kinases are about 40% identical and each contains a so-called pleckstrin homology domain. The 2.7 kb Bmx mRNA was expressed in endothelial cells and several human tissues by Northern blotting and an 80 kD Bmx polypeptide was detected in human endothelial cells. Immunoprecipitates of COS cells transfected with a Bmx expression vector and NIH3T3 cells expressing a Bmx retrovirus contained a tyrosyl phosphorylated Bmx polypeptide of similar molecular weight. The BMX gene was located in chromosomal band Xp22.2 between the DXS197 and DXS207 loci. Interestingly, chromosome X also contains the closest relative of BMX, the BTK gene, implicated in X-linked agammaglobulinemia. The BMX gene thus encodes a novel nonreceptor tyrosine kinase, which may play a role in the growth and differentiation of hematopoietic cells.     

Differential expression of ZAP-70 and Syk protein tyrosine kinases, and the role of this family of protein tyrosine kinases in TCR signaling

TCR stimulation results in the tyrosine phosphorylation of a number of cellular substrates. We have recently identified a 70-kDa protein tyrosine kinase, ZAP-70, which associates with the human TCR zeta-chain after TCR stimulation. We report here the isolation and sequence of a cDNA clone that encodes murine ZAP-70. Murine and human ZAP-70 share 93% amino acid identity and are homologous to the 72-kDa protein tyrosine kinase Syk. Syk has been implicated in the signal transduction pathways of the B cell membrane Ig and high affinity IgE receptors, Fc epsilon RI. In addition, we examined the tissue distribution of ZAP-70 and Syk in human and murine thymocyte subsets, B cells, and peripheral T cell subsets. ZAP-70 protein is expressed in all major thymocyte populations, with the level of expression being comparable to that found in both CD4+ and CD8+ peripheral T cells. Although Syk protein is also present in all thymocyte subsets, expression of Syk protein is down-regulated threefold to fourfold in peripheral T cells. In contrast to ZAP-70, expression of Syk is 12- to 15-fold higher in peripheral B cells when compared with peripheral T cells. In addition, whereas T cell stimulation results in down-regulation of Lck, no significant change in ZAP-70 or Syk protein is detected. Finally, we provide evidence that both ZAP-70 and Syk can associate with the TCR after TCR stimulation. With the use of a heterologous expression system, we show that, like ZAP-70, Syk is dependent upon a Src-family protein tyrosine kinase for association with the phosphorylated zeta-chain. Thus, the differential expression of these kinases suggests the possibility of different roles for ZAP-70 and Syk in TCR signaling and thymic development.     

Growth factors and insulin stimulate tyrosine phosphorylation of the 51C/SHIP2 protein

Antibodies raised against the 51C/SHIP2 inositol polyphosphate 5'-phosphatase were used to examine the effects of growth factors and insulin on the metabolism of this protein. Immunoblot analysis revealed that the 51C/SHIP2 protein was widely expressed in fibroblast and nonhematopoietic tumor cell lines, unlike the SHIP protein, which was found only in cell lines of hematopoietic origin. The 51C/SHIP2 antiserum precipitated a protein of approximately 145 kDa along with an activity which hydrolyzed phosphatidylinositol 3,4, 5-trisphosphate to phosphatidylinositol 3,4-bisphosphate. Tyrosine phosphorylation of the 51C/SHIP2 protein occurred in response to treatment of cells with epidermal growth (EGF), platelet-derived growth factor (PDGF), nerve growth factor (NGF), insulin-like growth factor-1 (IGF-1), or insulin. EGF and PDGF induced transient tyrosine phosphorylation of 51C/SHIP2, with maximal tyrosine phosphorylation occurring at 5-10 min following treatment and returning to near basal levels within 20 min. In contrast, treatment of cells with NGF, IGF-1, or insulin resulted in prolonged tyrosine phosphorylation of 51C/SHIP2 protein, with 40-80% maximal phosphorylation sustained for up to 2 h following agonist treatment. The kinetics of activation of the Akt/PKB protein kinase by the various factors correlated well with the kinetics of tyrosine phosphorylation of 51C/SHIP2. EGF, NGF, and PDGF stimulated the association of 51C/SHIP2 protein with the Shc adapter protein; however, no Shc could be detected in 51C/SHIP2-immune precipitates from cells treated with IGF-1 or insulin. The data suggest that 51C/SHIP2 may play a significant role in regulation of phosphatidylinositol 3'-kinase signaling by growth factors and insulin.     

Mona, a novel hematopoietic-specific adaptor interacting with the macrophage colony-stimulating factor receptor, is implicated in monocyte/macrophage development

The production, survival and function of monocytes and macrophages are regulated by the macrophage colony-stimulating factor (M-CSF or CSF-1) through its tyrosine kinase receptor Fms. Binding of M-CSF results in Fms autophosphorylation on specific tyrosines that act as docking sites for intracellular signaling molecules containing SH2 domains. Using a yeast two-hybrid screen, we cloned a novel adaptor protein which we called 'Mona' for monocytic adaptor. Mona contains one SH2 domain and two SH3 domains related to the Grb2 adaptor. Accordingly, Mona interacts with activated Fms on phosphorylated Tyr697, which is also the Grb2-binding site. Furthermore, Mona contains a unique proline-rich region located between the SH2 domain and the C-terminal SH3 domain, and is apparently devoid of any catalytic domain. Mona expression is restricted to two hematopoietic tissues: the spleen and the peripheral blood mononuclear cells, and is induced rapidly during monocytic differentiation of the myeloid NFS-60 cell line in response to M-CSF. Strikingly, overexpression of Mona in bone marrow cells results in strong reduction of M-CSF-dependent macrophage production in vitro. Taken together, our results suggest an important role for Mona in the regulation of monocyte/macrophage development as controlled by M-CSF.     

Characterization of a Schistosoma mansoni gene encoding a homologue of the Y-box binding protein

Hematopoietic stem cells (HSCs) support blood cells throughout life by utilizing their self-renewing and multilineage differentiating capabilities. Hematopoietic growth factors mediate their effects on stem cells by the tyrosine phosphorylation of proteins. Regulation of tyrosine phosphorylation is partially mediated by protein tyrosine phosphatases (PTPases). A possible mechanism by which hematopoietic stem cells maintain their self-renewing capacity and undifferentiated state is by controlling the balanced and opposing actions of protein tyrosine kinases (PTKs), receptors for growth factors, and PTPases. We have characterized the expression of PTPases in 5-fluorouracil (5-FU)-treated murine bone marrow cells, which represent a very primitive population of progenitors enriched for reconstituting stem cells, by using a consensus polymerase chain reaction (PCR) method. Several PTPases were expressed abundantly in the 5-FU-treated bone marrow stem cells. A novel PTP, termed protein tyrosine phosphatase receptor omicron (PTPRO), which is related to the homotypically adhering kappa, mu and PCP-2 receptor-type tyrosine phosphatases, was identified and characterized. We have cloned the murine and full-length human PTPRO cDNAs which share 89% homology, indicating that PTPRO is highly conserved between these species. The human PTPRO cDNA clone encodes a polypeptide of 1439 amino acids (aa) and has a calculated molecular mass of approximately 162 kDa. PTPRO consists of an extracellular segment containing a MAM domain, an immunoglobulin (Ig) domain, four fibronectin-type III (FN-III) repeats, a transmembrane segment, and two tandem intracellular PTP domains. The human PTPRO gene was assigned to human chromosome 1p35-pter using Southern blot analyses of genomic DNAs from rodent/human somatic hybrid cell lines containing human chromosome 1 or the p35-pter region of the chromosome. The mouse Ptpro gene was mapped to chromosome 4, closely linked to D4Mit16 and Elp1 (elliptocytosis-1), by using genomic DNAs from a (C57BL/6J x Mus spretus)F1 x Mus spretus backcross. In fetal tissues, PTPRO expression was observed in the brain and lung, whereas lower levels were observed in the kidney. In adult tissues, PTPRO was less restricted and was observed in the lung, heart, skeletal muscle, prostate, testis, and in various areas of the brain, indicating that PTPRO expression is developmentally regulated. Expression of PTPRO was also observed in human CD34+ bone marrow cells and 5-FU-treated murine primitive stem cells. These results suggest a potential role for PTPRO in stem cell adhesion and in mediating homophilic cell-cell interactions in other cell types.     

Identification of tyrosine residues within the intracellular domain of the erythropoietin receptor crucial for STAT5 activation

FDCP-1 cells are hematopoietic progenitor cells which require interleukin-3 for survival and proliferation. FDCP-1 cells stably transfected with the murine erythropoietin receptor cDNA survive and proliferate in the presence of erythropoietin. Erythropoietin induces the activation of the short forms (80 kDa) of STAT5 in the cells. Erythropoietin-induced activation of STAT5 was strongly reduced in cells expressing mutated variants of the erythropoietin receptors in which tyrosine residues in their intracellular domain have been eliminated. We determined that the erythropoietin receptor tyrosine residues 343 and 401 are independently necessary for STAT5 activation. The amino acid sequences surrounding these two tyrosine residues are very similar. Peptides comprising either phosphorylated Tyr343 or phosphorylated Tyr401, but not their unphosphorylated counterparts, inhibited the STAT5 activation. We propose that these two tyrosine residues of the erythropoietin receptor constitute docking sites for the STAT5 SH2 domain. The growth stimulus mediated by erythropoietin was decreased in cells expressing erythropoietin receptors lacking both Tyr343 and Tyr401. This suggests that STAT5 activation could be involved in the growth control of FDCP-1 cells.     

Retrovirus-mediated transfer of the human alpha-L-iduronidase cDNA into human hematopoietic progenitor cells leads to correction in trans of Hurler fibroblasts

Hurler syndrome (mucopolysaccharidosis IH or MPS IH) is a congenital mucopolysaccharide storage disorder resulting from a genetic deficiency of alpha-L-iduronidase (IDUA), which is required for lysosomal degradation of glycosaminoglycans heparan sulfate and dermatan sulfate. Even though histocompatible bone marrow transplantation has been applied for the treatment of Hurler syndrome, gene therapy via autologous bone marrow transplantation (BMT) may be more beneficial for this disease. Two retroviral vectors containing a full-length human IDUA cDNA were constructed using Moloney murine leukemia virus (MoMLV)-based vector backbones. High-titer vector-producing clones containing the L-HuID-SN and MFG-HuID retroviral vectors were established. The efficiency of gene transfer into primitive human CD34+ hematopoietic cells using both retroviral vectors is in the range of 18-23%. The level of enzyme expression in transduced primary bone marrow cells was increased 40- to 50-fold compared with that of sham-transduced cells. Enzyme produced by the progeny of the transduced human CD34+ cells carrying IDUA cDNA corrected Hurler fibroblasts via mannose-6-phosphate receptors. These findings suggest that genetically modified hematopoietic progenitor cells can potentially be useful for gene therapy of Hurler syndrome.     

Differential association of phosphatases with hematopoietic co-receptors bearing immunoreceptor tyrosine-based inhibition motifs

A novel family of inhibitory co-receptors has been recently defined according to the presence in their intracytoplasmic domain of immunoreceptor tyrosine-based inhibition motifs (ITIM). In particular, this family includes a low-affinity receptor for IgG, Fc gammaRIIB, which is widely expressed on hematopoietic cells, as well as killer cell inhibitory receptors (KIR) for major histocompatibility complex (MHC) class I proteins, expressed on both T and natural killer (NK) lymphocytes. Fc gammaRIIB and KIR inhibitory function depends upon the tyrosine phosphorylation of their respective ITIM. Phosphorylated Fc gammaRIIB and KIR ITIM bind the tandem SH2 tyrosine phosphatases, SHP-1 and SHP-2. Recently, Fc gammaRIIB has been shown to associate with a polyphosphate inositol 5-phosphatase, SHIP, which appears to be involved in its inhibitory function. Using cell lysate adsorption to phosphorylated ITIM peptides and surface plasmon resonance, we demonstrate here that, in contrast to Fc gammaRIIB, KIR (CD158b: p58.2) do not bind to SHIP, and only recruit SHP-1 and SHP-2. In addition, we show that point mutation of the amino acid residue in position tyrosine-2 of Fc gammaRIIB and KIR ITIM abolihes their binding to SHP-1 and SHP-2, but leaves intact the association of SHIP with Fc gammaRIIB ITIM. These data contribute to the structural definition of ITIM and document a differential recruitment of phosphatases by distinct ITIM. These findings also reveal that diverse strategies of inhibition are used by distinct members of the ITIM-bearing co-receptor family.     

Molecular cloning of a phosphotyrosine-independent ligand of the p56lck SH2 domain

A novel human cDNA encoding a cytosolic 62-kDa protein (p62) that binds to the Src homology 2 (SH2) domain of p56lck in a phosphotyrosine-independent manner has been cloned. The cDNA is composed of 2074 nucleotides with an open reading frame encoding 440 amino acids. Northern analysis suggests that p62 is expressed ubiquitously in all tissues examined. p62 is not homologous to any known protein in the data base. However, it contains a cysteine-rich region resembling a zinc finger motif, a potential G-protein-binding region, a PEST motif, and several potential phosphorylation sites. Using T7-epitope tagged p62 expression in HeLa cells, the expressed protein was shown to bind to the lck SH2 domain. Deletion of the N-terminal 50 amino acids abolished binding, but mutagenesis of the single tyrosine residue in this region had no effect on binding. Thus, the cloned cDNA indeed encodes the p62 protein, which is a phosphotyrosine-independent ligand for the lck SH2 domain. Its binding mechanism is unique with respect to binding modes of other known ligands for SH2 domains.     

Structural requirements for function of the Crkl adapter protein in fibroblasts and hematopoietic cells

Crkl is an adapter protein and phosphotyrosine-containing substrate implicated in transformation by the bcr-abl oncogene and in signaling by cytokines. When phosphorylated, Crkl binds through its Src homology 2 (SH2) domain to other tyrosine phosphoproteins such as paxillin and Cbl. Overexpression of Crkl in fibroblasts induces transformation. Here we examine the role of Crkl in hematopoietic cells and find that overexpression of Crkl confers a signal leading to increased adhesion to fibronectin. In both fibroblasts and hematopoietic cells, individual mutations or deletions of each SH2 and SH3 domain abrogated transformation and adhesion, respectively, indicating that interactions with other proteins such as Cbl and paxillin (SH2 domain) and Abl, Sos, and C3G (N-terminal SH3 domain) are essential for biological activity. In vivo and in vitro tryptic phosphopeptide mapping studies show that Crkl is phosphorylated on multiple tyrosine residues when overexpressed or when activated by Bcr-Abl. Mutation at tyrosine 207, a residue conserved in c-Crk, abrogates all in vivo tyrosine phosphorylation of Crkl. Despite this loss of phosphotyrosine, mutation at this site enhanced Crkl function as measured by complex formation with SH2 binding proteins, signal transduction to Jun Kinase, and fibroblast transformation. These observations implicate Crkl in cellular adhesion and demonstrate that Y207 functions as a negative regulatory site.     

Nuclear accumulation of G-actin in isolated rat hepatocytes by adenine nucleotides

Distinct inositol and phosphatidylinositol polyphosphates 5-phosphatases have recently been cloned. Primers have been designed coding for highly conserved amino acid regions that are shared between sequences of 5-phosphatases. One of the PCR fragment referred to as 51 C, shows 99% identity to a previously reported sequence (INPPL-1) present in the database. We report here the identification of cDNAs for a new SH2-domain-containing protein showing homology to the inositol 5-phosphatase SHIP and therefore referred to as SHIP2. SHIP2 differs at both N- and C-terminal ends with the sequence of INPPL-1. The translated sequence of SHIP2 encodes a 1258 amino acid protein with a predicted molecular mass of 142 kDa. Particularly high levels of SHIP2 were found in human heart, skeletal muscle and placenta as shown by Northern blot analysis. SHIP2 was also expressed in dog thyroid cells in primary culture where the expression was enhanced in TSH and EGF-stimulated cells.