Selective neural cell adhesion molecule signaling by Src family tyrosine kinases and tyrosine phosphatases

Nerve growth cone guidance is a highly complex feat, involving coordination of cell adhesion molecules, trophic factor gradients, and extracellular matrix proteins. While navigating through the developing nervous system, the growth cone must integrate diverse environmental signals into a singular response. The repertoire of growth cone responses to these extracellular cues includes axonal growth, fasciculation, and synaptic stabilization, which are achieved through dynamic changes in the cytoskeleton and modulation of gene expression. It has become evident that interactions between cell adhesion molecules can activate intracellular signaling pathways in neurons. Such signaling pathways are just beginning to be defined for the axonal growth promoting molecules L1 and NCAM which are members of the immunoglobulin (Ig) superfamily. Recent findings have revealed that L1 and NCAM induce neurite outgrowth by activating intracellular signaling pathways in the growth cone mediated by two different members of the src family of nonreceptor protein tyrosine kinases (PTKs), pp60(c-src) and p59(fyn5,6). Growth cones display diverse morphologies and variable motility on these different cell adhesion molecules, which are likely to be generated by src kinases. In this review we will address novel features of nonreceptor PTKs of the src family which dictate their distinctive molecular interactions with cell adhesion molecules and signaling components.     

rlk/TXK encodes two forms of a novel cysteine string tyrosine kinase activated by Src family kinases

Rlk/Txk is a member of the BTK/Tec family of tyrosine kinases and is primarily expressed in T lymphocytes. Unlike other members of this kinase family, Rlk lacks a pleckstrin homology (PH) domain near the amino terminus and instead contains a distinctive cysteine string motif. We demonstrate here that Rlk protein consists of two isoforms that arise by alternative initiation of translation from the same cDNA. The shorter, internally initiated protein species lacks the cysteine string motif and is located in the nucleus when expressed in the absence of the larger form. In contrast, the larger form is cytoplasmic. We show that the larger form is palmitoylated and that mutation of its cysteine string motif both abolishes palmitoylation and allows the protein to migrate to the nucleus. The cysteine string, therefore, is a critical determinant of both fatty acid modification and protein localization for the larger isoform of Rlk, suggesting that Rlk regulation is distinct from the other Btk family kinases. We further show that Rlk is phosphorylated and changes localization in response to T-cell-receptor (TCR) activation and, like the other Btk family kinases, can be phosphorylated and activated by Src family kinases. However, unlike the other Btk family members, Rlk is activated independently of the activity of phosphatidylinositol 3-kinase, consistent with its lack of a PH domain. Thus, Rlk has two distinct isoforms, each of which may have unique properties in signaling downstream from the TCR.     

Tyrosine phosphorylation-dependent and -independent associations of protein kinase C-delta with Src family kinases in the RBL-2H3 mast cell line: regulation of Src family kinase activity by protein kinase C-delta

Src kinases and protein kinase C (PKC) have been well studied for their role in oncogenic and normal cellular processes. Herein we report on a novel regulatory pathway mediated by the interaction of PKC-delta with p53/56Lsy (Lyn) and with p60Src (Src) that results in the phosphorylation and increased activity of Lyn and Src. In the RBL-2H3 mast cell line, the interaction of PKC-delta with Lyn required the activation of the high affinity receptor for IgE (FcsigmaRI) while the interaction with Src was constitutive. Increased complex formation of PKC-delta with Lyn or Src led to increased serine phosphorylation and activity of the Src family kinases. Conversely, Lyn was found to phosphorylate Lyn-associated and recombinant PKC-delta in vitro and the tyrosine 52 phosphorylated PKC-delta was recruited to associate with the Lyn SH2 domain. The constitutive association of PKC-delta with Src did not result in the tyrosine phosphorylation of PKC-delta prior to or after FsigmaRI engagement. However in cells over-expressing PKC-delta, FsigmaRI engagement resulted in the dramatic inhibition of Src activity and some inhibition of Lyn activity. Thus, the interaction and cross-talk of PKC-delta with Src family kinases suggests a novel and inter-dependent mechanism for regulation of enzymatic activity that may serve an important role in cellular responses.     

Solution structure of the SH3 domain from Bruton's tyrosine kinase

X-linked agammaglobulinemia (XLA) is a heritable immunodeficiency caused by mutations in the gene coding for Bruton's tyrosine kinase (Btk). Btk belongs to the Tec family of tyrosine kinases. Each member of the family contains five regions and mutations causing XLA have been isolated in all five regions. We have determined the solution structure of the Src homology 3 (SH3) domain of Btk using two- and three-dimensional nuclear magnetic resonance (NMR) spectroscopy on natural abundance and 15N-labeled protein material. The structure determination is complemented by investigation of backbone dynamics based on 15N NMR relaxation. The Btk SH3 forms a well-defined structure and shows the typical SH3 topology of two short antiparallel beta-sheets packed almost perpendicular to each other in a sandwich-like fold. The N- and C-termini are more flexible as are peptide fragments in the RT and n-Src loops. The studied Btk SH3 fragment adopts two slowly interconverting conformations with a relative concentration ratio of 7:1. The overall fold of the minor form is similar to that of the major form, as judged on the basis of observed NOE connectivities and small chemical shift differences. A tryptophan (W251) ring flip is the favored mechanism for interconversion, although other possibilities cannot be excluded. The side chain of Y223, which becomes autophosphorylated upon activation of Btk, is exposed within the potential SH3 ligand binding site. Finally, we compare the present Btk SH3 structure with other SH3 structures.     

Signaling pathways controlling trophoblast cell differentiation: Src family protein tyrosine kinases in the rat

A specific method for pancreatic elastase II activity analysis was developed. True elastase II activity could be discriminated from that of elastase I and chymotrypsin. The postnatal development of four pancreatic proteases in the duodenal juice of children and in the pancreatic homogenates of calves and piglets was measured. The study was carried out on patients without (14 children) and with (5 children) pancreatic insufficiency. Calves and piglets were either milk-fed or weaned until slaughter at different ages. Profiles of enzyme development were globally similar in milk-fed piglets and calves, while in children without pancreatic insufficiency, no significant change was observed between 4 and 168 months. In children with pancreatic insufficiency, enzyme activity was low. In animals, elastase II and chymotrypsin activities were maximal at birth, decreased with age, and probably were associated with the digestion of milk protein. In contrast, elastase I and trypsin activities increased markedly after weaning in connection with the intake of solid food.     

Regulation of Zap-70 by Src family tyrosine protein kinases in an antigen-specific T-cell line

To further understand the interactions between Zap-70, Src family kinases, and other T-cell proteins, we have examined the regulation of Zap-70 in the antigen-specific T-cell line BI-141. By analyzing derivatives containing an activated version of either p56lck or p59fynT, it was observed that the two Src-related enzymes augmented T-cell receptor (TCR)-mediated tyrosine phosphorylation of Zap-70, as well as its association with components of the antigen receptor complex. Importantly, the accumulation of TCR.Zap-70 complexes quantitatively and temporally correlated with the induction of tyrosine phosphorylation of the CD3 and zeta chains of TCR. Using a CD4-positive variant of BI-141, we also found that the ability of Zap-70 to undergo tyrosine phosphorylation and associate with TCR was enhanced by aggregation of TCR with the CD4 co-receptor. Further studies allowed the identification of two distinct pools of tyrosine-phosphorylated Zap-70 in activated T-cells. While one population was associated with TCR, the other was co-immunoprecipitated with a 120-kDa tyrosine-phosphorylated protein of unknown identity. In addition to supporting the notion that Src-related enzymes regulate the recruitment of Zap-70 in TCR signaling, these data added further complexity to previous models of regulation of Zap-70. Furthermore, they suggested that p120 may be an effector and/or a regulator of Zap-70 in activated T-lymphocytes.     

Identification of the Src family kinases, Lck and Fgr in platelets. Their tyrosine phosphorylation status and subcellular distribution compared with other Src family members

We have identified the Src family members, Lck and Fgr in resting human and rodent platelets and compared their subcellular distributions and tyrosine phosphorylation status to those of the other Src family kinases to gain insights into the signal transduction pathways active in maintaining platelets in the circulation. Like Fyn, Lyn, and Yes, most of Fgr and Lck was detergent-insoluble in human and rat platelets. In comparison, Src showed higher detergent solubility than the Src-related kinases. Most all human platelet Src was detergent-soluble, while that of rodent platelets was present in all detergent fractions. We also compared the tyrosine-phosphorylation status of Lck and Fgr to other Src family members in resting platelets using immunoprecipitation and immunoblotting. All of these Src family members except Fgr exhibited substantial phosphotyrosine antibody labeling. The partitioning of these kinases, with the exception of Src, with the detergent-insoluble fraction, their tyrosine-phosphorylation status, and co-localization with endocytotic vesicles lead us to hypothesize that the Src family kinases are involved in signaling events that drive cytoskeletal reorganization and active endocytosis of plasma proteins by circulating platelets.     

Bruton's tyrosine kinase regulates apoptosis and JNK/SAPK kinase activity

Mast cells derived from Bruton's tyrosine kinase (Btk)-defective xid or btk null mice showed greater expansion in culture containing interleukin-3 (IL-3) than those from wild-type (wt) mice. Although the proliferative response to IL-3 was not significantly different between the wt and xid mast cells, xid and btk null mast cells died by apoptosis more slowly than their wt counterparts upon IL-3 deprivation. Consistent with these findings, the apoptosis-linked c-Jun N-terminal kinase/stress-activated protein kinase (JNK) activity was compromised in these btk-mutated cells upon Fc(epsilon)RI crosslinking or upon stimulation with IL-3 or with stem cell factor. p38 activity was less severely, but significantly, affected by btk mutation, whereas extracellular signal-regulated kinases were not affected by the same mutation. Btk-mediated regulation of apoptosis and JNK activity was confirmed by reconstitution of btk null mutant mast cells with the wt btk cDNA. Furthermore, growth factor withdrawal induced the activation and sustained activity of JNK in wt mast cells, while JNK activity was consistently lower in btk-mutated mast cells. These results support the notion that Btk regulates apoptosis through the JNK activation.     

Protein tyrosine kinases Syk and ZAP-70 display distinct requirements for Src family kinases in immune response receptor signal transduction

Engagement of immunoreceptors in hemopoietic cells leads to activation of Src family tyrosine kinases as well as Syk or ZAP-70. Current models propose that Src family kinases are critical in immune response signal transduction through their role in phosphorylation of tyrosine residues within immunoreceptor tyrosine activation motifs (ITAMs; which recruit the SH2 domains of Syk or ZAP-70) and by direct phosphorylation of Syk and ZAP-70. Several lines of evidence suggest that Syk may not show the same dependence on activation by Src family kinases as ZAP-70. In this report, we used COS cells transiently transfected with components of the Fc epsilon RI complex (Lyn, Syk, and a chimeric CD8 receptor containing the cytoplasmic domain of the gamma subunit of Fc epsilon RI (CD8-gamma)) to examine the regulation of Syk activity. Syk was activated and phosphorylated in COS cells cotransfected with Lyn; however, in cells expressing CD8-gamma, activation of Syk and phosphorylation of CD8-gamma did not require coexpression of Lyn. Additional experiments indicate that gamma phosphorylation is dependent on Syk kinase activity and is independent of endogenous COS cell kinases. In parallel experiments, ZAP-70 was not activated by cotransfection with CD8-gamma, nor was CD8-gamma phosphorylated when coexpressed with ZAP-70 alone. Taken together, these studies indicate that Syk can be distinguished from ZAP-70 in its ability to be activated by coexpression with an ITAM-containing receptor without coexpression of a Src family kinase, and that Syk is capable of phosphorylating ITAM tyrosines under certain experimental conditions.     

Heat shock activates c-Src tyrosine kinases and phosphatidylinositol 3-kinase in NIH3T3 fibroblasts

There is increasing evidence that cellular responses to stress are in part regulated by protein kinases, although specific mechanisms are not well defined. The purpose of these experiments was to investigate potential upstream signaling events activated during heat shock in NIH3T3 fibroblasts. Experiments were designed to ask whether heat shock activates p60 c-Src tyrosine kinase or phosphatidylinositol 3-kinase (PI 3-kinase). Using in vitro protein kinase activity assays, it was demonstrated that heat shock stimulates c-Src and PI 3-kinase activity in a time-dependent manner. Also, there was increased PI 3-kinase activity in anti-phosphotyrosine and anti-c-Src immunoprecipitated immunocomplexes from heated cells. Heat shock activated mitogen-activated protein kinase (MAPK) and p70 S6 kinase (S6K) in these cells. The role of PI 3-kinase in regulating heat shock activation of MAPK and p70 S6K was investigated using wortmannin, a specific pharmacological inhibitor of PI 3-kinase. The results demonstrated that wortmannin inhibited heat shock activation of p70 S6K but only partially inhibited heat activation of MAPK. A dominant negative Raf mutant inhibited activation of MAPK by heat shock but did not inhibit heat shock stimulation of p70 S6K. Genistein, a tyrosine kinase inhibitor, and suramin, a growth factor receptor inhibitor, both inhibited heat shock stimulation of MAPK activity and tyrosine phosphorylation of MAPK. Furthermore, a selective epidermal growth factor receptor (EGFR) inhibitor, tryphostin AG1478, and a dominant negative EGFR mutant also inhibited heat shock activation of MAPK. Heat shock induced EGFR phosphorylation. These results suggest that early upstream signaling events in response to heat stress may involve activation of PI 3-kinase and tyrosine kinases, such as c-Src, and a growth factor receptor, such as EGFR; activation of important downstream pathways, such as MAPK and p70 S6K, occur by divergent signaling mechanisms similar to growth factor stimulation.     

Src family tyrosine kinase Lyn binds several proteins including paxillin in rat basophilic leukemia cells

Aggregation of the high affinity IgE receptors on rat basophilic leukemia (RBL-2H3) cells results in protein tyrosine phosphorylation although the receptor has no intrinsic enzymatic activity. The Src related protein tyrosine kinase p53/56lyn present in RBL-2H3 cells could play a role in this reaction. Here we have isolated the cDNA for rat Lyn and found it to be very homologous at the amino acid level to both the human and mouse proteins. A bacterially expressed maltose binding protein-Lyn (MBP-Lyn) fusion protein was already tyrosine phosphorylated and had tyrosine kinase activity. In a filter-binding assay, MBP-Lyn fusion protein (at 0.1 microM) specifically bound to several proteins of RBL-2H3 cells. In lysates of IgE receptor-activated cells, there was increased binding of MBP-Lyn to 65, 72, 78 and 110 kDa tyrosine phosphorylated proteins. The 72, 78 and 110 kDa tyrosine phosphorylated proteins were precipitated by a fusion protein containing the Lyn Src Homology 2 (SH2) domain. The 72 kDa Lyn binding protein was different from p72syk. Furthermore, paxillin, a cytoskeletal protein, was identified as one of the Lyn binding proteins. Thus Fc epsilon RI mediated signal transduction in RBL-2H3 cells may result from the interaction of p53/56lyn with paxillin, pp72, pp110 and other proteins.     

Mutation of unique region of Bruton's tyrosine kinase in immunodeficient XID mice

The cytoplasmic tyrosine kinase, Bruton's tyrosine kinase (Btk, formerly bpk or atk), is crucial for B cell development. Loss of kinase activity results in the human immunodeficiency, X-linked agammaglobulinemia, characterized by a failure to produce B cells. In the murine X-linked immunodeficiency (XID), B cells are present but respond abnormally to activating signals. The Btk gene, btk, was mapped to the xid region of the mouse X chromosome by interspecific backcross analysis. A single conserved residue within the amino terminal unique region of Btk was mutated in XID mice. This change in xid probably interferes with normal B cell signaling mediated by Btk protein interactions.     

A role for Bruton's tyrosine kinase (Btk) in platelet activation by collagen

Bruton's tyrosine kinase (Btk) is essential for normal B-cell receptor signalling. The lack of expression of functional Btk in humans leads to the B-cell deficiency X-linked agammaglobulinaemia (XLA). We report here that Btk is also important for signalling via the collagen receptor glycoprotein VI (GPVI) in platelets. GPVI is coupled to the Fc receptor gamma chain (FcRgamma). The FcRgamma-chain contains a consensus sequence known as the immune-receptor tyrosine-based activation motif (ITAM). Tyrosine phosphorylation of the ITAM upon GPVI stimulation is the initial step in the regulation of phospholipase C gamma2 (PLCgamma2) isoforms via the tyrosine kinase p72(Syk) (Syk) in platelets. Here we show that collagen and a collagen-related peptide (CRP), which binds to GPVI but does not bind to the integrin alpha2beta1, induced Btk tyrosine phosphorylation in platelets. Aggregation, dense granule secretion and calcium mobilisation were significantly diminished but not completely abolished in platelets from XLA patients in response to collagen and CRP. These effects were associated with a reduction in tyrosine phosphorylation of PLCgamma2. In contrast, aggregation and secretion stimulated by thrombin in Btk-deficient platelets were not significantly altered. Our results demonstrate that Btk is important for collagen signalling via GPVI, but is not essential for thrombin-mediated platelet activation.     

Protein-tyrosine phosphatase alpha regulates Src family kinases and alters cell-substratum adhesion

The roles of protein-tyrosine phosphatases (PTPs) in processes such as cell growth and adhesion are poorly understood. To explore the ability of specific PTPs to regulate cell signaling pathways initiated by stimulation of growth factor receptors, we expressed the receptor-like PTP, PTPalpha, in A431 epidermoid carcinoma cells. These cells express high levels of the epidermal growth factor (EGF) receptor and proliferate in response to the autocrine production of transforming growth factor-alpha. Conversely, EGF stimulation of A431 cells in vitro leads to growth inhibition and triggers the rapid detachment of these cells from the substratum. Although PTPalpha expression did not alter the growth characteristics of either unstimulated or EGF-stimulated cells, this phosphatase was associated with increased cell-substratum adhesion. Furthermore, PTPalpha-expressing A431 cells were strikingly resistant to EGF-induced cell rounding. Overexpression of PTPalpha in A431 cells was associated with the dephosphorylation/activation of specific Src family kinases, suggesting a potential mechanism for the observed alteration in A431 cell-substratum adhesion. Src kinase activation was dependent on the D1 catalytic subunit of PTPalpha, and there was evidence of association between PTPalpha and Src kinase(s). PTPalpha expression also led to increased association of Src kinase with the integrin-associated focal adhesion kinase, pp125(FAK). In addition, paxillin, a Src and/or pp125(FAK) substrate, displayed increased levels of tyrosine phosphorylation in PTPalpha-expressing cells and was associated with elevated amounts of Csk. In view of these alterations in focal adhesion-associated molecules in PTPalpha-expressing A431 cells, as well as the changes in adhesion demonstrated by these cells, we propose that PTPalpha may have a role in regulating cell-substratum adhesion.     

Fluoroaluminate induces activation and association of Src and Pyk2 tyrosine kinases in osteoblastic MC3T3-E1 cells

Fluoride is known to increase bone mass in vivo, probably through stimulation of osteoblast proliferation; however, the mechanisms of fluoroaluminate action in osteoblasts have not yet been elucidated. We have previously shown that in osteoblastic MC3T3-E1 cells, fluoroaluminate stimulates G protein-mediated protein tyrosine phosphorylation (Scaronuscarona, M., Standke, G. J. R., Jeschke, M., and Rohner, D. (1997) Biochem. Biophys. Res. Commun. 235, 680-684). Although the Ser/Thr kinases Erk1, Erk2, and p70(S6K) were activated in response to fluoroaluminate, the identity of fluoroaluminate-activated tyrosine kinase(s) remained elusive. In this study, we show that in MC3T3-E1 cells, fluoroaluminate induces a 110-kDa tyrosine-phosphorylated protein that we identify as Pyk2, a cytoplasmic tyrosine kinase related to Fak (focal adhesion kinase). The tyrosine phosphorylation of Pyk2 increased in a dose- and time-dependent manner. The autophosphorylation activity of Pyk2 increased 3-fold and reached its maximum within 10 min of fluoroaluminate treatment. Fluoroaluminate also induced activation of Src and the association of Pyk2 with Src. The phosphorylation of Src-associated Pyk2 increased >20-fold in in vitro kinase assays, suggesting that Pyk2 is phosphorylated by Src. Although MC3T3-E1 cells express much more Fak than Pyk2, Src preferentially associated with Pyk2. In vitro, Pyk2 bound to the Src SH2 domain, suggesting that this interaction mediates the Src-Pyk2 association in cells. These data indicate that osteoblastic cells express Pyk2, which is tyrosine-phosphorylated and activated in response to G protein activation by fluoroaluminate, and that the mechanism of Pyk2 activation most likely involves Src. Thus, Src and Pyk2 are tyrosine kinases involved in G protein-mediated tyrosine phosphorylation in osteoblastic cells and may be important for the osteogenic action of fluoroaluminate.