Differentiation and survival of neuronal cell types requires the action of neurotrophic polypeptides such as nerve growth factor (NGF). In the central and peripheral nervous system and the phaeochromocytoma cell model PC12, NGF exerts its effects through the activation of the signalling capacity of Trk, a receptor tyrosine kinase (RTK) which upon interaction with NGF becomes phosphorylated on tyrosines and thereby acquires the potential to interact with signal‐transducing proteins such as phospholipase C‐gamma (PLC gamma), phosphatidylinositol‐3′‐kinase (PI3′‐K) and SHC. Mutagenesis of the specific binding sites for these src homology 2 (SH2) domain‐containing substrates within the Trk cytoplasmic domain suggests a non‐essential function of PI3′‐K and reveals a major role for the signal controlled by the SHC binding site at tyrosine 490 and a co‐operative function of the PLC gamma‐mediated pathway for neuronal differentiation of PC12 cells.

Alternative pre-mRNA splicing patterns can change an extracellular stimulus, but the signaling pathways leading to these changes are still poorly characterized. Here, we describe a tyrosinephosphorylated nuclear protein, YT521-B, and show that it interacts with the nuclear transcriptosomal component scaffold attachment factor B, and the 68-kDa Src substrate associated during mitosis, Sam68. Northern blot analysis demonstrated ubiquitous expression, but detailed RNA in situ analysis revealed cell type specificity in the brain. YT521-B protein is localized in the nucleoplasm and concentrated in 5-20 large nuclear dots. Deletion analysis demonstrated that the formation of these dots depends on the presence of the amino-terminal glutamic acid-rich domain and the carboxyl-terminal glutamic acid/arginine-rich region. We show that the latter comprises an important protein-protein interaction domain. The Src family kinase p59 fyn -mediated tyrosine phosphorylation of Sam68 negatively regulates its association with YT521-B, and overexpression of p59 fyn dissolves nuclear dots containing YT521-B. In vivo splicing assays demonstrated that YT521-B modulates alternative splice site selection in a concentration-dependent manner. Together, our data indicate that YT521-B and Sam68 may be part of a signal transduction pathway that influences splice site selection. INTRODUCTIONThe members of the signal transduction and activation of RNA (STAR) or GRP33/SAM68/GLD1 domain-containing protein family are proposed mediators connecting signal transduction pathways and RNA metabolism (Vernet and Artzt, 1997). The prototype of these proteins is Sam68, the 68-kDa Src substrate associated during mitosis (Taylor and Shalloway, 1994). Sam68 and its related protein family members contain an RNA binding domain that has been referred to as the STAR domain (Vernet and Artzt, 1997), GSG (GRP33/SAM68/GLD1) (Jones et al.), or SGQ (SAM68/ GLD1/Quaking homology domain) (Lin et al., 1997). This domain contains an RNA binding KH domain module flanked by two Qua1 and Qua2 domains that are required for high-affinity RNA binding. In addition, Sam68 contains an RGG box that has been implicated in RNA binding (Kiledjian and Dreyfuss, 1992), as well as proline-and tyrosine-rich regions involved in binding to Src homology 3 (SH3) and SH2 domains. Sam68 thus associates with a variety of signaling molecules, including members of the Src family tyrosine kinases, growth factor receptor-bound protein 2 (GRB-2), and phospholipase C␥-1 . The RNA binding ability and oligomerization of Sam68 are inhibited by p59 fyn (Chen et al., 1997), a member of the Src family of kinases. These data suggest that Sam68 functions as a multifunctional SH3 and SH2 adapter protein with the ability to link cytosolic signaling pathways to downstream effects involved in RNA metabolism, such as alternative splicing.Alternative splicing is an important mechanism for creating different protein isoforms from a single gene. In many cases, stop codons are introduced by alternative splicing, whic...

Interaction of the nerve growth factor (NGF) receptor/Trk with cellular substrates was investigated by transient co‐overexpression in human 293 fibroblasts using ET‐R, a chimeric receptor consisting of the epidermal growth factor receptor (EGF‐R) extracellular ligand binding domain and the Trk transmembrane and intracellular signal‐generating sequences. The chimera was fully functional, and associated with and phosphorylated phospholipase C gamma (PLC gamma), ras GTPase‐activating protein (GAP) and the non‐catalytic subunit of phosphatidylinositol‐3′‐kinase, p85, in a ligand‐dependent manner. Deletion of 15 C‐terminal amino acids, including tyrosine 785 (Y‐785) abrogated receptor and substrate phosphorylation activities. Mutation of Y‐785 to phenylalanine somewhat impaired receptor phosphorylation activity, which was reflected in reduced GAP and p85 phosphorylation. In contrast, ET‐YF phosphorylation of PLC gamma was significantly reduced, while the high affinity association potential with this substrate was abrogated by this point mutation in vitro and in intact cells. Furthermore, a tyrosine‐phosphorylated synthetic C‐terminal peptide competitively inhibited Trk cytoplasmic domain association with PLC gamma. Thus, the short C‐terminal tail appears to be a crucial structural element of the Trk cytoplasmic domain, and phosphorylated Y‐785 is a major and selective interaction site for PLC gamma.

An orally bioavailable and blood-brain barrier penetrating analog of the kinase inhibitor K252a was able to prevent the typical motor deficits in the tau (P301L) transgenic mouse model (JNPL3) and markedly reduce soluble aggregated hyperphosphorylated tau. However, neurofibrillary tangle counts were not reduced in the successfully treated cohort, suggesting that the main cytotoxic effects of tau are not exerted by neurofibrillary tangles but by lower molecular mass aggregates of tau. Our findings strongly suggest that abnormal tau hyperphosphorylation plays a critical role in the development of tauopathy and suggest a previously undescribed treatment strategy for neurodegenerative diseases involving tau pathology.Alzheimer's disease ͉ extracellular signal-regulated kinase inhibitor ͉ paired helical filament ͉ tangles

The serine/threonine kinase p21-activated kinase (PAK) has been implicated as a downstream effector of the small GTPases Rac and Cdc42. While these GTPases evidently induce a variety of morphological changes, the role(s) of PAK remains elusive. Here we report that overexpression of βPAK in PC12 cells induces a Rac phenotype, including cell spreading/ membrane ruffling, and increased lamellipodia formation at growth cones and shafts of nerve growth factor-induced neurites. These effects are still observed in cells expressing kinase-negative or Rac/ Cdc42 binding-deficient PAK mutants, indicating that kinase-and p21-binding domains are not involved. Furthermore, lamellipodia formation in all cell lines, including those expressing Rac binding-deficient PAK, is inhibited significantly by dominant-negative RacN17. Equal inhibition is achieved by blocking PAK interaction with the guanine nucleotide exchange factor PIX using a specific N-terminal PAK fragment. We conclude that PAK, via its N-terminal non-catalytic domain, acts upstream of Rac mediating lamellipodia formation through interaction with PIX.

Shc is an adaptor protein that contains two phosphotyrosine-binding domains, a Src homology 2 (SH2) domain and the newly described phosphotyrosine interaction (PI) domain. Shc interacts with several tyrosine-phosphorylated proteins and is itself tyrosine-phosphorylated in cells stimulated with a variety of growth factors and cytokines. Upon phosphorylation, Shc binds to the Grb2.Sos complex leading to the activation of the Ras signaling pathway. Mutational analysis of the nerve growth factor (NGF) receptor (TrkA) suggested that the binding of Shc to the activated receptor is required for NGF-induced neuronal differentiation of PC12 cells. Here we report that the PI domain of Shc directly binds to tyrosine 490 on the autophosphorylated NGF receptor. The PI domain specifically recognizes an I/LXN-PXpY motif (where p indicates phosphorylation) as determined by phosphopeptide competition assay. In addition, the PI domain is able to efficiently compete for binding of full-length Shc proteins to the NGF receptor. In PC12 cells, the Shc SH2 domain interacts with an unidentified tyrosine-phosphorylated protein of 115 kDa but not with the activated NGF receptor. The ability of Shc to interact with different tyrosine-phosphorylated proteins via its PI and SH2 domains may allow Shc to play a unique role in tyrosine kinase signal transduction pathways.

Abstract. Receptor tyrosine kinases play essential roles in morphogenesis and differentiation of epithelia. Here we examined various tyrosine kinase receptors, which are preferentially expressed in epithelia (c-met, c-ros, c-neu, and the keratin growth factor [KGF] receptor), for their capacity to induce cell motility and branching morphogenesis of epithelial cells. We exchanged the ligand-binding domain of these receptors by the ectodomain of trkA and could thus control signaling by the new ligand, NGF. We demonstrate here that the tyrosine kinases of c-met, c-ros, c-neu, the KGF receptor, and trkA, but not the insulin receptor, induced scattering and increased motility of kidney epithelial cells in tissue culture. Mutational analysis suggests that SHC binding is essential for scattering and increased cell motility induced by trkA. The induction of motility in epithelial cells is thus an important feature of various receptor tyrosine kinases, which in vivo play a role in embryogenesis and metastasis. In contrast, only the c-met receptor promoted branching morphogenesis of kidney epithelial ceils in three-dimensional matrices, which resemble the formation of tubular epithelia in development. Interestingly, the ability of c-met to induce morphogenesis could be transferred to trkA, when in a novel receptor hybrid COOH-terminal sequences of c-met (including Y14 to Y16) were fused to the trkA kinase domain. These data demonstrate that tubulogenesis of epithelia is a restricted activity of tyrosine kinases, as yet only demonstrated for the c-met receptor. We predict the existence of specific substrates that mediate this morphogenesis signal.

-EJB 95 1044/1 PC12 cells, which lack platelet derived-growth-factor (PDGF) receptors, have been stably transfected with a chimaera consisting of the extracellular domain of the p-PDGF receptor and the intracellular and transmembrane domains of the nerve-growth-factor receptor Trk-A (termed PT-R). Mutation of the Trk-A residue Tyr490 to phenylalanine prevents the association with Shc, while similar mutations at Tyr751 or Tyr785 are reported to prevent interaction of Trk-A with the p85 subunit of inositol phospholipid 3-kinase and phospholipase C-yl , respectively. The strong and sustained activation of p42 and p44 mitogenactivated-protein kinases induced by PDGF-B/B in PC12/PT-R cells was unaffected by mutation of Tyr78.5 or Tyr751 to phenylalanine, but was smaller and transient after mutation of Tyr490, and almost abolished by the double mutation of Tyr490 and Tyr78.5. Mutation of Tyr490 reduced by 70% the PDGFinduced increase in inositol phospholipid 3-kinase activity immunoprecipitated from cell extracts with antiphosphotyrosine monoclonal antibodies and greatly suppressed the PDGF-induced increase in the intracellular products of inositol phospholipid 3-kinase, while mutation of Tyr751 or Tyr785 had no effect. Mutation of Tyr785 (but not mutation of Tyr490 or Tyr7.51) abolished PDGF-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate. Mutation of Tyr490, alone or in combination with mutation of Tyr751 and Tyr785, had no effect on the PDGF-induced activation of p70 S6 kinase (~7 0 "~) .However, the activation of p70ShK by PDGF (or nerve growth factor), but not the activation of mitogen-activatedprotein kinase, was prevented by two structurally unrelated inhibitors of inositol phospholipid 3-kinase, wortmannin or LY294002. Our results demonstrate the following : (1) the phosphorylation of Tyr490 plays a major role in the activation of inositol phospholipid 3-kinase and formation of 3-phosphorylated inositol lipids and confirm that the phosphorylation of Tyr 785 triggers the activation of phospholipase C-yl in vivo. (2) Tyr490 phosphorylation (but not inositol phospholipid 3-kinase activation) is also required for strong and sustained activation of mitogen-activated-protein kinase and neuronal differentiation, while the smaller and more transient activation of mitogen-activated-protein kinase, produced by the activation of phospholipase C-yl is insufficient to trigger the neuronal differentiation of PT-R cells. (3) Inositol phospholipid 3-kinase is required for the activation of p70SfiK, but only a small increase in inositol phospholipid 3-kinase activity and the level of 3-phosphorylated inositol lipids is required for maximal p70SbK activation.Keywords: PC12 cells ; nerve growth factor; platelet-derived growth factor; inositol phospholipid 3-kinase ; mitogen-activated-protein kinase.Nerve growth factor (NGF), a neurotrophic factor essential for the survival and development of sympathetic neurones, causes PC12 cells to differentiate from immature chromaffin cells to a sympathetic neuron-like phenoty...