Neurotrophins are key regulators of the fate and shape of neuronal cells and act as guidance cues for growth cones by remodeling the actin cytoskeleton. Actin dynamics is controlled by Rho GTPases. We identified a novel Rho GTPase-activating protein (Grit) for Rho/Rac/Cdc42 small GTPases. Grit was abundant in neuronal cells and directly interacted with TrkA, a high-affinity receptor for nerve growth factor (NGF). Another pool of Grit was recruited to the activated receptor tyrosine kinase through its binding to N-Shc and CrkL/Crk, adapter molecules downstream of activated receptor tyrosine kinases. Overexpression of the TrkA-binding region of Grit inhibited NGF-induced neurite elongation. Further, we found some tendency for neurite promotion in full-length Grit-overexpressing PC12 cells upon NGF stimulation. These results suggest that Grit, a novel TrkA-interacting protein, regulates neurite outgrowth by modulating the Rho family of small GTPases.

The phosphotyrosine (pTyr) adapter Shc/ShcA is a major connector in various tyrosine kinase signalings following a variety of stimulation such as growth factor/ neurotrophin, as well as in those following calcium in¯ux and integrin activation. As in other tissues, Shc has been implicated in neuronal signalings; however, recent evidence suggests that N-Shc/ShcC and Sck/ShcB would take over most of the roles of Shc in mature central neurons, and switching phenomena between Shc and NShc expression were observed in several neuronal paradigms. Little is, however, known as to the signaloutput dierences between Shc and N-Shc. Here we determined the ecacy of Shc and N-Shc toward Erk activation in NGF-treated PC12 cells, and found that NShc transduced Grb2/Sos/Ras-dependent Erk activation less eciently than Shc. This was mainly because N-Shc has only one high-anity Grb2-binding site, whereas Shc has two such sites. Phosphopeptide mapping revealed that N-Shc has novel tyrosine-phosphorylation sites at Y259/Y260 and Y286; in vivo-phosphorylation of these tyrosines was demonstrated by site-speci®c anti-pTyr antibodies. Phosphorylated Y286 bound to several proteins, of which one was Crk. The pY221/pY222 site, corresponding to one of the Grb2-binding sites of Shc, also preferentially bound to Crk. The phosphorylationdependent interaction between N-Shc and Crk was demonstrated in vitro and in vivo. These results indicate that N-Shc has speci®c features of signal-output, and further suggest that the switching between Shc and NShc during neural development and regeneration would lead to dierentiation of downstream signalings.

We have identified a novel gene, designated KRAP (Ki-ras-induced actin-interacting protein), encoding a protein of 1,259 amino acids with coiled-coil regions and transmembrane regions, from the cDNA library of human colon cancer HCT116 cells, as one of the genes upregulated by activated Ki-ras. While KRAP was rarely expressed in normal colon epithelium, deregulated constitutive KRAP expression was observed in some other colon cancer cells. In normal tissues, KRAP was strongly expressed in pancreas and testis. Anti-KRAP polyclonal antibodies detected endogenous KRAP as the molecular size of M r 180,000, and immunofluorescence microscopy and cytochalasin E treatment revealed that KRAP was clearly associated with the actin filaments. Furthermore, KRAP was localized as a membrane-bound form with extracellular regions. These results together suggested KRAP might be involved in the regulation of filamentous actin and signals from the outside of the cells.