The Crk-associated substrate (Cas) is a unique docking protein that possesses a repetitive stretch of tyrosine-containing motifs and an Src homology 3 (SH3) domain. Embryonic fibroblasts lacking Cas demonstrated resistance to Src-induced transformation along with impaired actin bundling and cell motility, indicating critical roles of Cas in actin cytoskeleton organization, cell migration, and oncogenesis. To gain further insight into roles of each domain of Cas in these processes, a compensation assay was performed by expressing a series of Cas mutants in Cas-deficient fibroblasts. The results showed that motifs containing YDxP were indispensable for actin cytoskeleton organization and cell migration, suggesting that CrkII-mediated signaling regulates these biological processes. The C-terminal Src-binding domain played essential roles in cell migration and membrane localization of Cas, although it was dispensable in the organization of actin stress fibers. Furthermore, the Src-binding domain was also a prerequisite for Src transformation possibly, because of its crucial role in the phosphorylation of Cas during transformation. Overall, differential uses of the Cas domains in individual biological processes were demonstrated. Cas1 docking protein was initially identified and cloned as a major phosphotyrosine-containing protein in cells transformed by v-src and v-crk oncogenes (1, 2). It has a structure with a number of protein-protein interaction domains, including an N-terminal Src homology 3 (SH3) domain, a substrate domain (SD) that consists of a cluster of YxxP motifs (one YLVP, four YQxPs, nine YDxPs, and one YAVP), and a C-terminal Srcbinding domain containing motifs YDYV (amino acids 762-765) and RPLPSPP (2). The substrate domain offers docking sites for several molecules including adaptor proteins Crk, Nck, and an inositol 5Ј-phosphatase, SHIP2 (SH2-containing inositol 5Ј-phosphatase), through their SH2 domains in a phosphorylation dependent manner (3-5). Motifs RPLPSPP and YDYV in the SB domain serve as direct binding sites for SH3 and SH2

The amounts of normal and compensatory polyamines of polyamine-requirnmg Escherichia coli mutants grown in the absence of polyamines were determined. Although aminopropylcadaverine, a compensatory polyamine, was synthesized by MA135 (speB) and DR112 (speA speB), no aminopropylcadaverine or only small amounts of aminopropylcadaverine were synthesized by EWH319 (speA speB speC speD) and MA261 (speB speC), respectively. The average mass doubling times of MA135, DR112, MA261, and EWH319 grown in the absence of polyamines were 113, 105, 260, and 318 min, respectively. The correlation of these values with the sum of spermidine plus aminopropylcadaverine suggested that aminopropylcadaverine is important for cell growth in the presence of limiting amounts of normal polyamines. This hypothesis is supported by the results of aminopropylcadaverine stimulation of the in vitro synthesis of polyphenylalanine and MS2 RNA replicase and of its stimulation of the growth of MA261. For the following reasons, it was concluded that aminopropylcadaverine was synthesized preferentially from cadaverine made by ornithine decarboxylase: (i) aminopropylcadaverine was synthesized in relatively large amounts in cells (MA135 and DR112) which possess ornithine decarboxylase; (ii) ornithine decarboxylase catalyzed the decarboxylation of lysine in vitro, and (iii) the in vivo formation of aminopropylcadaverine was inhibited by an inhibitor of ornithine decarboxylase.

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1 We examined regeneration of endothelial cells (ECs), neointima formation, decreased endotheliumdependent relaxation (EDR) and changes in the contents of L-arginine, NG-monomethyl-L-arginine (L-NMMA), asymmetrical NG, N0-dimethylarginine (ADMA) and symmetrical NG,NG-dimethylarginine (SDMA) in the regnerated ECs, 6 weeks after balloon denudation of the rabbit carotid artery. 2 Regeneration of ECs was completed in 6 weeks and a significant neointima formation accompanied by the decreased EDR was observed. 3 L-NMMA and ADMA contents in the regenerated ECs (23.5 + 4.3 and 21.2 + 2.0 pmol mg-' DNA, respectively) were significantly (P <0.05 and P <0.01) higher than those in the control ECs (8.8 + 3.0 and 7.4 + 1.9 pmol mg'l DNA, respectively), whereas L-arginine was significantly (P < 0.005) decreased in the regenerated ECs (31,470+1,050 pmol mg-' DNA) as compared to that in the control ECs (47,870+ 1,890 pmol mg-' DNA). SDMA content was below the assay limits. 4 L-NMMA and ADMA, but not SDMA, inhibited the EDR induced by acetylcholine in a concentration-dependent manner. The inhibition with L-NMMA and ADMA was prevented by an addition of L-arginine, but not by D-arginine. 5 These results suggest that the accumulation of endogenous inhibitors for nitric oxide synthesis and decreased L-arginine content are associated with decreased NO production/release from regenerated ECs and neointima formation. Keywords: Neointima formation; endogenous inhibitors for NO synthesis; balloon denudation; decreased endotheliumdependent relaxation; EDRF/NO Introduction MethodsAs proposed by Ross & Glomset (1976a,b) and Ross (1986), denudation and nondenudation intimal injuries may play an important role in the development of atherosclerotic vascular disease (Ip et al., 1990). We have reported that restoration of the endothelium after balloon denudation of the rabbit carotid artery was completed in 6 weeks and that regenerated ECs were less capable of producing/releasing the endothelium-derived relaxing factor (EDRF/nitric oxide: NO) (Azuma et al., 1990;Niimi et al., 1994). EDRF/NO inhibits platelet adhesion and aggregation (Azuma et al., 1986;Radomski et al., 1987), which is considered as an initiating event for neointima formation (Ross, 1986;Schwartz et al., 1981). It has also been demonstrated that NO inhibits smooth muscle cell proliferation (Garg & Hassid, 1989). These findings when considered together suggest that the neointima formation after endothelial removal might be caused at least partly through impairment of the ability of ECs to produce/release EDRF/NO. However, the mechanism by which the production/release of EDRF/NO is reduced remains unclear. Recently, Vallance et al. (1992) have obtained evidence NG-monomethyl-L-arginine (L-NMMA) and asymmetrical NG,NG-dimethyl arginine (ADMA) play a role as endogenous inhibitors of NO synthesis. The present experiments were, therefore, designed to determine the regeneration of ECs, the neointima formation, the decreased EDR and the changes in content of L-arginine, L-NMMA, ADMA an...

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