Biomedical subjects

L M Delmolino

Publications and source records attributed to L M Delmolino.

3 recordsLinked to original sources

PubMed2001

Multiple mechanisms regulate subcellular localization of human CDC6.

CDC6 is a protein essential for DNA replication, the expression and abundance of which are cell cycle-regulated in Saccharomyces cerevisiae. We have demonstrated previously that the subcellular localization of the human CDC6 homolog, HsCDC6, is cell cycle-dependent: nuclear during G(1) phase and cytoplasmic during S phase. Here we demonstrate that endogenous HsCDC6 is phosphorylated during the G(1)/S transition. The N-terminal region contains putative cyclin-dependent kinase phosphorylation sites adjoining nuclear localization sequences (NLSs) and a cyclin-docking motif, whereas the C-terminal region contains a nuclear export signal (NES). In addition, we show that the observed regulated subcellular localization depends on phosphorylation status, NLS, and NES. When the four putative substrate sites (serines 45, 54, 74, and 106) for cyclin-dependent kinases are mutated to alanines, the resulting HsCDC6A4 protein is localized predominantly to the nucleus. This localization depends upon two functional NLSs, because expression of HsCDC6 containing mutations in the two putative NLSs results in predominantly cytoplasmic distribution. Furthermore, mutation of the four serines to phosphate-mimicking aspartates (HsCDC6D4) results in strictly cytoplasmic localization. This cytoplasmic localization depends upon the C-terminal NES. Together these results demonstrate that HsCDC6 is phosphorylated at the G(1)/S phase of the cell cycle and that the phosphorylation status determines the subcellular localization.

Amino Acid Sequence↗

PubMed2001

COP-1, a member of the CCN family, is a heparin-induced growth arrest specific gene in vascular smooth muscle cells.

Vascular smooth muscle cell (VSMC) hyperplasia is responsible for the failure of 15-30% of vascular surgical procedures such as coronary artery bypass grafts and angioplasties. We and others have shown that heparin suppresses VSMC proliferation in vivo and in cell culture. We hypothesize that heparin inhibits VSMC proliferation by binding to cell surface receptors, resulting in selective modulation of mitogenic signal transduction pathways and altered transcription of a specific subset of growth regulatory genes. To test this idea, we used subtractive hybridization to identify differentially expressed mRNAs in heparin-treated and untreated VSMC. We identified a heparin induced mRNA identical to Cop-1, a member of the CCN family of proteins which are secreted, cysteine-rich modular proteins involved in growth regulation and migration. Cop-1 from smooth muscle cells appears to have a different expression pattern and possibly different functions than Cop-1 from other cells. Cop-1 mRNA is expressed at high levels in quiescent VSMC and at low levels in proliferating VSMC, an expression pattern highly characteristic of growth arrest specific genes. Cop-1 mRNA is expressed at high levels in heparin treated VSMC and COP-1 protein is secreted into culture medium. In tissues, Cop-1 expression is observed in the uninjured rat aorta suggesting a possible role for Cop-1 in vivo. We found PDGF, but not EGF, inhibits the expression of Cop-1 in VSMC. Neither TGF-beta nor interferon-beta, two inhibitors of VSMC proliferation, were able to induce Cop-1 expression. In addition, heparin does not induce Cop-1 mRNA in endothelial cells and VSMC resistant to the antiproliferative effect of heparin. Conditioned medium from cells over-expressing COP-1 protein inhibits VSMC proliferation in culture. Together, our data indicate that COP-1 may play a role in the antiproliferative mechanism of action of heparin.

Amino Acid Sequence↗

PubMed1997

Heparin suppresses sgk, an early response gene in proliferating vascular smooth muscle cells.

Vascular smooth muscle cell (VSMC) hyperplasia plays a central role in chronic and acute vascular pathology including arteriosclerosis and restenosis following vascular surgery. The glycosaminoglycans of the heparan sulfate class, including heparin, inhibit VSMC proliferation in animals and in culture. Heparin binds to high affinity sites on the cell surface, selectively modulates mitogenic signal transduction pathway(s), and rapidly alters transcription of several genes. To further explore the molecular mechanisms responsible for this growth inhibition, we have employed the differential display technique to identify heparin-regulated genes. Here we demonstrate that heparin inhibits the expression of the early response gene sgk (serum and glucocorticoid-regulated kinase). The expression of sgk is not inhibited by chondroitin sulfate, a nonantiproliferative glycosaminoglycan, suggesting that sgk suppression may play a functional role in the antiproliferative effect of heparin. This idea is strengthened by the finding that heparin does not inhibit sgk expression in VSMCs resistant to the antiproliferative effect of heparin or in vascular endothelial cells which are unresponsive to heparin. Expression of sgk mRNA diminishes with increasing concentrations of heparin. Finally, sgk expression is not suppressed by other growth inhibitors such as transforming growth factor-beta 1 (TGF-beta 1) and interferon-beta (IFN-beta), suggesting separate and distinct effects of these growth inhibitors on the mitogenic pathway.

Animals↗