Activation of Stat3 in endothelial cells following hypoxia-reoxygenation is mediated by Rac1 and protein Kinase C.

Stat3 is an important transcription factor that regulates both proinflammatory and anit-apoptotic pathways in the heart. This study examined the mechanisms of activation of Stat3 in human endothelial cells following hypoxia/reoxygenation (H/R). By expression of constitutively active Rac1 mutant protein, and by RNA silencing of Rac1, we found that Stat3 forms a multiprotein complex with Rac1 and PKC in an H/R-dependent manner, which at least in part, appears to regulate Stat3 S727 phosphorylation. Selective inhibition of PKC with calphostin C produces a marked suppression of Stat3 S727 phosphorylation. The association of Stat3 with Rax1 occurs predominantly at the cell membrane, but also inside the nucleus, and occurs through the binding of the coiled-coil domain of Stat3 to the 54 NH(2)-terminal residues of Rac1. Transfection with a peptide comprising the NH(2)-terminal 17 amino acid residues of Rac1-dependent signaling pathways resulting in physical association between Rac1 and Stat3 and the formation of a novel multiprotein complex with PKC.

Inhibition of protein 4.1 R and NuMA interaction by mutagenization of their binding-sites abrogates nuclear localization of 4.1 R.

Protein 4.1R(4.1R) is a multifunctional structural protein recently implicated in nuclear assembly and cell division. We earlier demonstrated that 4.1R forms a multiprotein complex with mitotic spindle and spindle pole organizing proteins, such as NuMA, dynein, and dynactin, by binding to residues 1788-1810 of NuMA through amino acids encoded by exons 20 and 21 in 24 kD domain. Employing random-and site-directed mutagenesis combined with glycine- and alanine-scanning, we have identified amino acids of 4.1 R and NuMA that sustain their interaction, and have analyzed the effect of mutating the binding sites on their intracellular colocalization. We found that V762, V765, and V767 of 4.1 R, and 11800, 11801,11803, Tl 804, and M1805 of NuMA are necessary for their interaction. GST-fusion peptides of the 4.1R24 kD domain bound to residues 1785-2115 of NuMA in in vitro binding assays, but the binding was inhibited by alanine substitutions of V762, V765, and V767 of 4.1 R, or residues 1800-1805 of NuMA. Additionally, expression of variants of 4.1 R or NuMA that inhibit their in vitro binding also abrogated nuclear localization of 4.1 Rand colocalization with NuMA. Our findings suggest a crucial role of 4.1 R/NuMA interaction in localization and function of 4.1 R in the nucleus.

Carcinogenic chromium(VI)-induced protein oxidation and lipid peroxidation: implications in DNA-protein crosslinking.

Hexavalent chromium [Cr(VI)] compounds are Group-I human carcinogens. Cr(VI)-induced DNA-protein crosslinks (DPCs) have been implicated in the mutagenic and carcinogenic effects of Cr(VI). Although multiple mechanisms have been suggested for Cr(VI)-induced DNA-protein crosslinking, the mechanism of formation of DNA-protein crosslinks is not well understood. In this study, we explored the hypothesis that Cr(VI)-induced DPCs could be formed via generation of protein carbonyls and malonaldehyde (MDA) through protein oxidation and lipid peroxidation, respectively. Treatment of human leukemic T-lymphocyte MOLT4 cells with potassium chromate induced the formation of protein carbonyls and DPCs within 2 h, but increased the level of MDA only after 4 h, in a dose-dependent manner. Chromate treatment of MOLT4 cell homogenates also resulted in increased formation of MDA and protein carbonyls in a dose-dependent manner. EPR spectrometry in combination with spin trapping techniques revealed that reaction of Cr(VI) with biological reductants such as NADPH, glutathione reductase or H(2)O(2) generates Cr(V) and (*)OH radicals. Pretreatment of cells with antioxidants such as alpha-tocopherol or Tiron inhibited chromate-induced increase in formation of protein carbonyls, MDA and DPCs, but pretreatment of cells with riboflavin or 3-aminotriazole, a catalase inhibitor, had the opposite effect. Our results, for the first time, demonstrate that Cr(VI) exposure increases the cellular level of protein carbonyls and that Cr(VI)-induced DPCs may be formed, at least in part, via generation of protein carbonyls.

TIMAP, a novel CAAX box protein regulated by TGF-beta1 and expressed in endothelial cells.

Representational difference analysis of the glomerular endothelial cell response to transforming growth factor-beta1 (TGF-beta1) revealed a novel gene, TIMAP (TGF-beta-inhibited membrane-associated protein), which contains 10 exons and maps to human chromosome 20.q11.22. By Northern blot, TIMAP mRNA is highly expressed in all cultured endothelial and hematopoietic cells. The frequency of the TIMAP SAGE tag is much greater in endothelial cell SAGE databases than in nonendothelial cells. Immunofluorescence studies of rat tissues show that anti-TIMAP antibodies localize to vascular endothelium. TGF-beta1 represses TIMAP through a protein synthesis- and histone deacetylase-dependent process. The TIMAP protein contains five ankyrin repeats, a protein phosphatase-1 (PP1)-interacting domain, a COOH-terminal CAAX box, a domain arrangement similar to that of MYPT3, and a PP1 inhibitor. A green fluorescent protein-TIMAP fusion protein localized to the plasma membrane in a CAAX box-dependent fashion. Hence, TIMAP is a novel gene highly expressed in endothelial and hematopoietic cells and regulated by TGF-beta1. On the basis of its domain structure, TIMAP may serve a signaling function, potentially through interaction with PP1.