More than thirty small guanine nucleotide-binding proteins related to the ras-encoded oncoprotein, termed Ras or p21ras, are known. They regulate many fundamental processes in all eukaryotic cells, such as growth, vesicle traffic and cytoskeletal organization. GTPase-activating proteins (GAPs) accelerate the intrinsic rate of GTP hydrolysis of Ras-related proteins, leading to down-regulation of the active GTP-bound form. For p21ras, two GAP proteins are known, rasGAP and the neurofibromatosis (NF1) gene product. There is evidence that rasGAP may also be a target protein for regulation by Ras and be involved in downstream signalling. We have purified a GAP protein for p21rho, which is involved in the regulation of the actin cytoskeleton. Partial sequencing of rhoGAP reveals significant homology with the product of the bcr (breakpoint cluster region) gene, the translocation breakpoint in Philadelphia chromosome-positive chronic myeloid leukaemias. We show here that the carboxy-terminal domains of the bcr-encoded protein (Bcr) and of a Bcr-related protein, n-chimaerin, are both GAP proteins for the Ras-related GTP-binding protein, p21rac. This result suggest that Bcr could be a target for regulation by Rac and has important new implications for the role of bcr translocations in leukaemia.

Cdc42Hs is involved in cytoskeletal reorganization and is required for neurite outgrowth in N1E-115 cells. To investigate the molecular mechanism by which Cdc42Hs regulates these processes, a search for novel Cdc42Hs protein partners was undertaken by yeast two-hybrid assay. Here, we identify the 58-kD substrate of the insulin receptor tyrosine kinase (IRS-58) as a Cdc42Hs target. IRS-58 is a brain-enriched protein comprising at least four protein–protein interaction sites: a Cdc42Hs binding site, an Src homology (SH)3-binding site, an SH3 domain, and a tryptophan, tyrptophan (WW)-binding domain. Expression of IRS-58 in Swiss 3T3 cells leads to reorganization of the filamentous (F)-actin cytoskeleton, involving loss of stress fibers and formation of filopodia and clusters. In N1E-115 cells IRS-58 induces neurite outgrowth with high complexity. Expression of a deletion mutant of IRS-58, which lacks the SH3- and WW-binding domains, induced neurite extension without complexity in N1E-115 cells. In Swiss 3T3 cells and N1E-115 cells, IRS-58 colocalizes with F-actin in clusters and filopodia. An IRS-581267N mutant unable to bind Cdc42Hs failed to localize with F-actin to induce neurite outgrowth or significant cytoskeletal reorganization. These results suggest that Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing protein complexes via adaptor proteins such as IRS-58 to F-actin.

Neurite outgrowth is influenced by positive and negative signals that include the semaphorins, an important family of axonal outgrowth inhibitors. Here we report that the Rac GTPase activating protein (GAP) ␣2-chimaerin is involved in Semaphorin 3A (Sema 3A) signaling. In dorsal root ganglion neurons, Sema 3A-induced growth cone collapse was inhibited by ␣2-chimaerin mutated to eliminate GAP activity or interaction with phosphotyrosine. Activation of ␣2-chimaerin by phorbol ester caused growth cone collapse. Active ␣2-chimaerin interacts with collapsin response mediator protein-2 (CRMP-2) and cyclin-dependent kinase (Cdk) 5/p35 kinase through its SH2 and GAP domains, respectively. Cdk5 phosphorylates CRMP-2 at serine 522, possibly facilitating phosphorylation of serine 518 and threonine 514 by glycogen synthase kinase 3␤ (GSK3␤), a kinase previously implicated in Sema 3A signaling. Phosphorylation of CRMP-2 serine 522 was essential for Sema 3A-induced growth cone collapse, which is dependent on Cdk5 but not Rho kinase activity. ␣2-chimaerin, like CRMP-2, can associate with the Sema 3A receptor. These results indicate that active ␣2-chimaerin Rac GAP, Cdk5/p35, and its substrate CRMP-2, are implicated in the dynamics of growth cone guidance initiated through Sema 3A signaling.

The human heat-shock protein multigene family comprises several highly conserved proteins with structural and functional properties in common, but which vary in the extent of their inducibility in response to metabolic stress. We have isolated and characterized a novel human HSP70 cDNA, HSP70B' cDNA, and its corresponding gene sequence. HSP70B' cDNA hybrid-selected an mRNA encoding a more basic 70 kDa heat-shock protein that both the major stress-inducible HSP70 and constitutively expressed HSC70 heat-shock proteins, which in common with other heat-shock 70 kDa proteins bound ATP. The complete HSP70B' gene was sequenced and, like the major inducible HSP70 gene, is devoid of introns. The HSP70B' gene has 77% sequence similarity to the HSP70 gene and 70% similarity to HSC70 cDNA, with greatest sequence divergence towards the 3'-terminus. The HSP70B' gene represents a functional gene, as indicated by Northern-blot analysis with specific oligonucleotides, hybrid-selected translation with a specific 3' cDNA sequence and S1 nuclease protection experiments. In contrast with HSP70 mRNA, which is present at low concentrations in HeLa cells and readily induced by heat or CdCl2 treatment in both fibroblasts and HeLa cells, HSP70B' mRNA was induced only at higher temperature and showed no basal expression. The differences in patterns of induction may be due to the special features of the promoter region of the HSP70B' gene.

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Neuronal differentiation involves Rac and Cdc42 GTPases. ␣-Chimaerin, a Rac/Cdc42 regulator, occurs as ␣1-and alternatively spliced Src homology 2 (SH2) domain-containing ␣2-isoforms. ␣2-chimaerin mRNA was highly expressed in the rat embryonic nervous system, especially in early postmitotic neurons. ␣1-chimaerin mRNA was undetectable before embryonic day 16.5. Adult ␣2-chimaerin mRNA was restricted to neurons within specific brain regions, with highest expression in the entorhinal cortex. ␣2-chimaerin protein localized to neuronal perikarya, dendrites, and axons. The overall pattern of ␣2-chimaerin mRNA expression resembles that of cyclindependent kinase regulator p35 (CDK5/p35) which participates in neuronal differentiation and with which chimaerin interacts. To determine whether ␣2-chimaerin may have a role in neuronal differentiation and the relevance of the SH2 domain, the morphological effects of both chimaerin isoforms were investigated in N1E-115 neuroblastoma cells. When plated on poly-lysine, transient ␣2-chimaerin but not ␣1-chimaerin transfectants formed neurites. Permanent ␣2-chimaerin transfectants generated neurites whether or not they were stimulated by serum starvation, and many cells were enlarged. Permanent ␣1-chimaerin transfectants displayed numerous microspikes and contained F-actin clusters, a Cdc42-phenotype, but generated few neurites. In neuroblastoma cells, ␣2-chimaerin was predominantly soluble with some being membrane-associated, whereas ␣1-chimaerin was absent from the cytosol, being membrane-and cytoskeleton-associated, paralleling their subcellular distribution in brain. Transient transfection with ␣2-chimaerin mutated in the SH2 domain (N94H) generated an ␣1-chimaerin-like phenotype, protein partitioned in the particulate fraction, and in NGF-stimulated pheochromocytoma cell line 12 (PC12) cells, neurite formation was inhibited. These results indicate a role for ␣2-chimaerin in morphological differentiation for which its SH2 domain is vital.

Carboxypeptidase E (CPE), which cleaves C-terminal amino acid residues and is involved in neuropeptide processing, is itself subject to intracellular processing. Human CPE cDNA was isolated and sequence comparisons were made with those of a previously isolated brain cDNA (M1622) encoding rat CPE and of other human carboxypeptidases (M and N). Human (2.5 kb) and rat (2.1 kb) CPE cDNAs approximated to the size of their respective mRNAs; additional sequences were located in putative 5' and 3' untranslated regions of human CPE mRNA. There is 79% sequence similarity between human and rat CPE cDNAs, with greater similarity (89%) over the coding region and short sections of the non-coding sequence. The predicted 476-amino acid-residue sequences of human and rat preproCPEs are highly conserved (96% identity), with lower degree of similarity of the N-terminal signal peptide (76%). Human CPE showed 51% and 43% sequence similarity to human CPN and CPM respectively, with discrete regions of divergence dispersed between the highly conserved mechanistically implicated regions. Antiserum generated from a fusion protein, synthesized in Escherichia coli from constructs of the human cDNA, recognized an approx. 50 kDa membrane protein and a smaller soluble protein in rat and human brain preparations, corresponding to the two forms of native CPE. Human CPE mRNA transcripts directed the synthesis in reticulocyte lysate of a 54 kDa translation product, which in the presence of dog pancreas microsomal membranes was co-translationally processed with cleavage, insertion into membranes and glycosylation. Three processed forms were generated, the largest (56 kDa) and smallest (52 kDa) being equally glycosylated. The membrane association of the processed translation products and of native brain membrane CPE, detected immunologically, was resistant to moderate alkali but not pH 11.5 extraction. These results are consistent with secondary-structure predictions that CPE is a peripheral membrane protein. The dissimilar regions of human carboxypeptidases may provide information on sequences responsible for their different cellular disposition.

A human brain-specific cDNA encoding n-chimaerin, a protein of predicted molecular mass 34 kDa, has sequence identity with two different proteins: protein kinase C (PKC) at the N-terminus and BCR protein [product of the breakpointcluster-region (BCR) gene, involved in Philadelphia chromosome translocation] at the C-terminus [Hall, Monfries, Smith, Lim, Kozma, Ahmed, Vannaisungham, Leung & Lim (1990) J. Mol. Biol. 211,[11][12][13][14][15][16]. The sequence identity of nchimaerin with PKC includes the cysteine-rich motif CX2CXl3CX2CX7CX7C, and amino acids upstream of the first cysteine residue, but not the kinase domain. This region of PKC has been implicated in the binding of diacylglycerol and phorbol esters in a phospholipid-dependent fashion. Part of this cysteine-rich motif (CX2CX13CX2C) has the potential of forming a 'Zn-finger' structure. Phorbol esters cause a variety of physiological changes and are among the most potent tumour promoters that have been described. PKC is the only known protein targdt for these compounds. We now report that n-chimaerin cDNA encodes a novel phospholipid-dependent phorbol ester receptor, with the cysteine-rich region being responsible for this activity. This finding has wide implications for previous studies equating phorbol ester (Hall et al., 1990). The mRNA has a neuronal distribution. n-Chimaerin cDNA encodes a protein of predicted molecular mass 34 kDa. The protein sequence of human nchimaerin cDNA has no direct counterpart in the PIR (Protein Identification Resource) (DNAstar computer search; Lipman & Pearson, 1985) database. The N-terminus contains a stretch of 60 amino acids rich in cysteine residues, with 48 % similarity to one half (Cl b) of a duplicated sequence found in the Cl region of protein kinase C (PKC) Hall et al., 1990). Immediately adjacent to this region there is 42% sequence similarity to the C-terminus of BCR protein-[product of the breakpoint-cluster-region (BCR) gene involved in Philadelphia (Ph') chromosome translocation (Heisterkamp et al., 1985;Hall et al., 1990) (Diamond et al., 1980;Blumberg, 1980;Leach et al., 1983). The only known protein target for phorbol esters is PKC, an enzyme involved in the modulation of ion channels and receptors, gene expression and cell proliferation (Neidel et al., 1983;Nishizuka, 1984Nishizuka, , 1988 S. Ahmed and others RR1 transformed with pATH23 (TrpE, 37 kDa), pATCMl (TrpE-n-chimaerin fusion, 70 kDa), pATCM2 (TrpE-N-terminal n-chimaerin fusion, 50 kDa) or pATCM3 (TrpE/C-terminal n-chimaerin, 55 kDa) were grown overnight in M9 minimal salts with 1 mM-MgSO4/0.l mM-CaCl2/vitamin B-I (100 ,ug/ml)/ tryptophan (40 #sg/ml)/ampicillin (100 l,g/ml) or in LB broth with ampicillin. The overnight cultures were diluted 10-fold into 100 ml of the minimal-salts medium, and 3,8-indoleacrylic acid (20 ,ug/ml) was added when the cultures reached an A550 of 0.4. Cells were grown for a further 2-3 h, harvested and then resuspended in 3 ml of 20 mM-Tris/HCl (pH 7.5)/0.25 M-sucrose/2 mM-EDTA/IO mM-EGTA/leupeptin (20 jsg/ml)/0.5...