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The regulation of morphological changes in eukaryotic cells is a complex process involving major components of the cytoskeleton including actin microfilaments, microtubules, and intermediate filaments (IFs). The putative effector of RhoA, RhoA-binding kinase alpha (ROKalpha), is a serine/threonine kinase that has been implicated in the reorganization of actin filaments and in myosin contractility. Here, we show that ROKalpha also directly affects the structural integrity of IFs. Overexpression of active ROKalpha, like that of RhoA, caused the collapse of filamentous vimentin, a type III IF. A RhoA-binding-deficient, kinase-inactive ROKalpha inhibited the collapse of vimentin IFs induced by RhoA in HeLa cells. In vitro, ROKalpha bound and phosphorylated vimentin at its head-rod domain, thereby inhibiting the assembly of vimentin. ROKalpha colocalized predominantly with the filamentous vimentin network, which remained intact in serum-starved cells. Treatment of cells with vinblastine, a microtubule-disrupting agent, also resulted in filamentous vimentin collapse and concomitant ROKalpha translocation to the cell periphery. ROKalpha translocation did not occur when the vimentin network remained intact in vinblastine-treated cells at 4 degreesC or in the presence of the dominant-negative RhoAN19 mutant. Transient translocation of ROKalpha was also observed in cells subjected to heat shock, which caused the disassembly of the vimentin network. Thus, the translocation of ROKalpha to the cell periphery upon overexpression of RhoAV14 or growth factor treatment is associated with disassembly of vimentin IFs. These results indicate that Rho effectors known to act on microfilaments may be involved in regulating the assembly of IFs. Vimentin when phosphorylated also exhibits reduced affinity for the inactive ROKalpha. The translocation of ROKalpha from IFs to the cell periphery upon action by activated RhoA and ROKalpha suggests that ROKalpha may initiate its own cascade of activation.

n-Chimerin (alpha 1-chimerin) is a brain GTPase-activating protein (GAP) for the ras-related p21rac. We now report the occurrence of another form of chimerin, termed alpha 2-chimerin. This is the product of an alternately spliced transcript of the human n-chimerin gene encoding an N-terminal SH2 (src homology 2) domain in addition to the phorbol ester receptor and GAP domains. alpha 1- and alpha 2-chimerin mRNAs were expressed differently. In the rat brain, only alpha 1-chimerin mRNA was expressed in cerebellar Purkinje cells, although both alpha 1- and alpha 2-chimerin mRNAs occurred in neurons in the cerebral cortex, hippocampus, and thalamus. Only alpha 2-chimerin RNA was expressed in rat testes, in early pachytene spermatocytes. A 45-kDa SH2-containing chimerin corresponding to the alpha 2 form was purified from rat brain. As with Escherichia coli 45-kDa recombinant alpha 2-chimerin, purified brain alpha 2-chimerin exhibited racGAP activity which was stimulated by phosphatidylserine. The recombinant SH2 domain bound several 32P-labelled phosphoproteins of PC12 cells, whose phosphorylation increased in response to trophic factors, including nerve growth factor. To examine the relationships of alpha 1- and alpha 2-chimerin transcripts, human genomic DNA clones were characterized. In alpha 2-chimerin mRNA, a 3' splice acceptor site within exon 1 of alpha 1-chimerin mRNA was used, replacing its 5' untranslated region and N-terminal coding sequence. The single human n-chimerin gene was mapped to chromosome 2q31-q32.1, colocalizing with the CRE-BP1 transcription factor gene (2q32). It contained several splice junctions conserved with the sequence-related protein kinase C and bcr genes. alpha 2-Chimerin is only the second SH2-containing GAP and the first example of an SH2 domain generated by alternate splicing.