Changes in cellular growth and dramatic alterations in cell morphology and adhesion are common features of cells transformed by oncogenic protein tyrosine kinases, such as pp6O0 and other members of the Src family. In this report, we present evidence for the stable association of two Src family kinases (pp60 and pp59fiy) with tyrosine-phosphorylated forms of a focal adhesion-associated protein tyrosine kinase, pp125FAK. In Src-transformed chicken embryo cells, most of the ppl25FAK was stably complexed with activated pp60" (e.g., pp60527F). The stable association of pp25FAK with pp6O 121 in vivo required the structural integrity of the Src SH2 domain. The association of pp60W27F and ppl25FAK could be reconstituted in vitro by incubation of normal cell extracts with glutathione S-transferase fusion proteins containing SH2 or SH3/SH2 domains of pp6Olr'. Furthermore, the association of isolated SH2 or SH3/SH2 domains with in vitro 32P-labeled pp125FAK protected the major site of pp125FAK autophosphorylation from digestion with a tyrosine phosphatase, indicating that the autophosphorylation site of ppl25FAK participates in binding with Src. Immunoprecipitation of Src family kinases from extracts of normal chicken embryo cells revealed stable complexes of pp590' and tyrosine-phosphorylated ppl25FAK. These data provide evidence for a direct interaction between two cytoplasmic nonreceptor protein tyrosine kinases and suggest that Src may contribute to changes in pp125FAK regulation in transformed cells. Furthermore, ppl25FAK may directly participate in the targeting of pp59f" or possibly other Src family kinases to focal adhesions in normal cells.Oncogenic protein tyrosine kinases, such as pp6Osrc and other members of the Src family, induce overt changes in cellular growth and dramatic alterations in cell morphology and adhesion (reviewed in references 5, 18, and 27). For Src, these changes stem from the pp6Q'-induced tyrosine phosphorylation of specific cellular proteins, many of which are associated with elements of the cytoskeletal network (12,20,27,33,45). The functional interaction of pp6Q" with its cellular substrates is regulated at several levels. The tyrosine kinase activity of the cellular homolog of Src, pp6ocsrC, is negatively regulated by the phosphorylation of a C-terminal tyrosine residue (Tyr-527) (6-8). An alteration of Tyr-527 (for example, the mutation of Tyr-527 to either Phe or Ser) results in elevated tyrosine kinase activity and oncogenic transformation (3,22,32,36). The negative regulation of pp6OcsP' kinase activity also requires the interaction of phosphorylated Tyr-527 with an amino-terminal domain of Src termed SH2 (src homology 2) (4, 37). The SH2 domain is a highly conserved sequence motif that is present in many cellular proteins, some of which are involved in regulating events in signal transduction (reviewed in reference 31), and that directs the high-affinity binding of phosphotyrosinecontaining peptide sequences (43 phosphorylated proteins and contributes to intramolecular interactions...

No abstract

Western blot analysis of neuronal tissues taken from fear-conditioned rats showed a selective activation of phosphatidylinositol 3-kinase (PI-3 kinase) in the amygdala. PI-3 kinase was also activated in response to long-term potentiation (LTP)-inducing tetanic stimulation. PI-3 kinase inhibitors blocked tetanus-induced LTP as well as PI-3 kinase activation. In parallel, these inhibitors interfered with long-term fear memory while leaving short-term memory intact. Tetanus and forskolin-induced activation of mitogen-activated protein kinase (MAPK) was blocked by PI-3 kinase inhibitors, which also inhibited cAMP response element binding protein (CREB) phosphorylation. These results provide novel evidence of a requirement of PI-3 kinase activation in the amygdala for synaptic plasticity and memory consolidation, and this activation may occur at a point upstream of MAPK activation.

Transformation of chicken embryo cells by oncogenic forms of pp64Yrc (e.g., pp60V" or The Rous sarcoma virus transforming gene product, pp6Ovsrc, is both necessary and sufficient for transformation (reviewed in reference 33). The transforming capacity of pp60 is dependent upon activation of tyrosine kinase catalytic function, its association with cell membranes (7), and its ability to stably associate with cellular proteins (22). Regulation of the tyrosine kinase activity is accomplished by phosphorylation of a carboxyl-terminal tyrosine residue, Tyr-527, a residue that is missing from pp6ovsrc (51). In pp6OCSrCs, mutation of Y-527 to phenylalanine (pp60527F) results in a transformation-competent form of pp60 that displays an elevated tyrosine-specific kinase activity both in vitro and in vivo (5,25,36,40). Membrane association of Src proteins requires the myristylation of a glycine residue at amino acid position 2. Mutations that change this glycine residue to alanine prevent myristylation and membrane association and block the transforming capacity of pp6ovsrc and pp60527F (2-4, 19, 39, 46 with another tyrosine-phosphorylated cellular protein, ppl3O) forms a stable complex with activated pp6Orc (22,38). The stable association of AFAP-110 in vivo requires the structural integrity of the SH3 and SH2 (Src homology 3 and 2) domains of pp60Yrc. The SH3 and SH2 domains are conserved not only among the Src family of nonreceptor tyrosine kinases (26) but also among a number of proteins known to participate in signal transduction, e.g., the GTPase-activating protein (52, 53), phospholipase C--y (50), phosphatidylinositol 3-kinase (17, 24, 31), and the oncoprotein pp47-Crk (30).A monoclonal antibody (MAb 4C3) specific for AFAP-110 has been used to characterize the intracellular localization of AFAP-llO, the steady-state level of phosphorylation, and its association with pp60Orc (21, 22). AFAP-110 is associated with actin stress fibers in normal CE cells and, upon transformation by pp6Orc, undergoes a striking rearrangement in cellular localization into rosette-like structures referred to as podosomes (8,22). The redistribution of AFAP-110 in Srctransformed cells is similar to that observed for actin filaments and other cytoskeleton-associated proteins (1,11,22). In Src-transformed CE cells, AFAP-110 is tyrosine phosphorylated and forms stable complexes with activated forms of pp6Q"rc. Tyrosine phosphorylation of AFAP-110 and its association with pp6o0rc are not observed in cells infected with transformation-defective variants of pp6Q"rc that contain deletions within the SH3 or SH2 domain (22). The stable association between AFAP-110 and pp6Orc correlates with the fully transformed phenotype of Src-transformed cells. 7892on May 11, 2018 by guest

Utilizing an in vivo labeling method on synchronized cultures, we have previously defined a 28-kilobase (kb) replication initiation locus in the amplified dihydrofolate reductase domain of a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) (N. H. Heintz and J. L. Hamlin, Proc. Natl. Acad. Sci. USA 79: 4083-4087, 1982; N. H. Heintz and J. L. Hamlin, Biochemistry 22:3552-3557, 1983; N. H. Heintz, J. D. Milbrandt, K. S. Greisen, and J. L. Hamlin, Nature [London] 302:439-441, 1983). To locate the origin of replication in this 243-kb amplicon with more precision, we used an in-gel renaturation procedure (I. Roninson, Nucleic Acids Res. 11:5413-5431, 1983) to examine the labeling pattern of restriction fragments from the amplicon in the early S phase. This method eliminates background labeling from single-copy sequences and allows quantitation of the relative radioactivity in individual fragments. We used this procedure to follow the movement of replication forks through the amplicons, to roughly localize the initiation locus, and to estimate the rate of fork travel. We also used a slight modification of this method (termed hybridization enhancement) to illuminate the labeling pattern of smaller restriction fragments derived solely from the initiation locus itself, thereby increasing resolution. Our preliminary results suggest that there are actually two distinct initiation sites in the amplicon that are separated by -22 kb.

Memory extinction refers to a gradual decrease of the previously acquired response when exposed to conditional stimulus without pairing with unconditional stimulus. Here we show for the first time that fear training-induced phosphorylation of specific substrates in the rat amygdala is reduced after extinction trials and is accompanied by an increase in the protein level and enzymatic activity of calcineurin. In parallel, calcineurin inhibitors prevented extinction-induced protein dephosphorylation as well as extinction of fear memory. Thus, extinction training increased phosphatase activity likely via an expression of calcineurin. Calcineurin then created a negative-feedback loop and directly or indirectly dephosphorylated specific substrates, which, in their phosphorylated state, were required for memory consolidation. Accordingly, in our experimental condition, extinction could be ascribed at least in part to a weakening of the original signaling.

Mammalian Sterile 20-like kinase 3 (Mst3), the physiological functions of which are unknown, is a member of the germinal center kinase-III family. It contains a conserved kinase domain at its NH 2 terminus, whereas there is a regulatory domain at its COOH terminus. In this study we demonstrate that endogenous Mst3 is specifically cleaved when Jurkat cells were treated with anti-Fas antibody or staurosporine and that this cleavage is inhibited by the caspase inhibitor, Ac-DEVD-CHO. Using apoptotic Jurkat cell extracts and recombinant caspases, we mapped the caspase cleavage site, AETD 313 , which is at the junction of the NH 2 -terminal kinase domain and the COOH-terminal regulatory domain. Caspase-mediated cleavage of Mst3 activates its intrinsic kinase activity, suggesting that the COOH-terminal domain of Mst3 negatively regulates the kinase domain. Furthermore, proteolytic removal of the Mst3 COOH-terminal domain by caspases promotes nuclear translocation. Ectopic expression of either wild-type or COOH-terminal truncated Mst3 in cells results in DNA fragmentation and morphological changes characteristic of apoptosis. By contrast, no such changes were exhibited for catalytically inactive Mst3, implicating the involvement of Mst3 kinase activity for mediation of these effects. Collectively, these results support the notion that caspase-mediated proteolytic activation of Mst3 contributes to apoptosis.

MST3 is a member of the sterile-20 protein kinase family with a unique preference for manganese ion as a cofactor in vitro; however, its biological function is largely unknown. Suppression of endogenous MST3 by small interference RNA enhanced cellular migration in MCF-7 cells with reduced expression of E-cadherin at the edge of migrating cells. The alteration of cellular migration and protruding can be rescued by RNA interference-resistant MST3. The expression of surface integrin and Golgi apparatus was not altered, but phosphorylation on tyrosine 118 and tyrosine 31 of paxillin was attenuated by MST3 small interfering RNA (siRNA). Threonine 178 was determined to be one of the two main autophosphorylation sites of MST3 in vitro. Mutant T178A MST3, containing alanine instead of threonine at codon 178, lost autophosphorylation and kinase activities. Overexpression of wild type MST3, but not the T178A mutant MST3, inhibited migration and spreading in Madin-Darby canine kidney cells. MST3 could phosphorylate the protein-tyrosine phosphatase (PTP)-PEST and inhibit the tyrosine phosphatase activity of PTP-PEST. We conclude that MST3 inhibits cell migration in a fashion dependent on autophosphorylation and may regulate paxillin phosphorylation through tyrosine phosphatase PTP-PEST.