Biomedical subjects

L S Ulsh

Publications and source records attributed to L S Ulsh.

5 recordsLinked to original sources

PubMed1988

Novel phosphorylation of c-ras p21 by protein kinases.

Novel p21 phosphorylation was found in cells expressing high levels of this product of c-H- and c-K-ras genes. Phorbol 12,13-dibutyrate, a protein kinase C (PKC) activator, and permeable c-AMP derivatives, which activate protein kinase A (PKA), stimulated phosphorylation of K-ras(4B) p21 in 416B cells 3 to 5 fold. By tryptic peptide mapping, it was found that both PKC and PKA phosphorylated in vitro the K-ras p21 at the same site as was found in p21 from cells labeled with [32P]orthophosphate in vivo. A common site of H-ras p21 was also phosphorylated by both PKC and PKA, although phosphopeptides of H-ras p21 were distinct from those of K-ras p21. The construction of a mutant by site-directed mutagenesis allowed the identification of serine-177 as the phosphorylation site of H-ras p21. This novel phosphorylation site lies in the hypervariable region, which links the globular catalytic domain of p21 to the membrane-anchoring site at the C-terminus, a location suggesting that this phosphorylation plays a role in modulating transmembrane signaling.

Amino Acid Sequence↗

PubMed1986

Structure and function of p21 ras proteins.

Cancer is a malfunction of cellular growth control. The discovery of oncogenes, first in transforming retroviruses, and later in human and animal tumors, may have uncovered the key to understanding one of the most elusive subjects of basic cell biology, namely, the controlling mechanisms of cell growth. The ras gene family encodes a group of closely related 21,000 dalton (p21) proteins with special affinity for guanine nucleotides. Other cellular proteins with similar biochemical properties, collectively known as G-proteins, include the regulatory G proteins of adenylate cyclase, the alpha subunit of transducin of retina rod outer segments, the recently identified rho gene proteins, and perhaps also the elongation factors, EF-Tu and EF-G, of the protein synthesis system. These G-proteins have roles in cellular signal transduction; by analogy p21 may have a similar cellular function in mediating the flow of growth control signals. Recent progress in the cloning and sequencing of these genes, overproduction of gene products in E. coli, protein engineering, detailed biochemical characterization, and the molecular structure determined by high resolution X-ray crystallography, have helped to elucidate in great detail the structure and function of p21 ras proteins. p21 appears to have a small membrane binding domain at the C-terminus, which contains a palmitylation site at cysteine-186, four amino acid residues from the end. Separated by a variable "hinge" region, most of the rest of ras amino acid sequences are highly conserved in nature. Four regions of extensive sequence homology among G-proteins constitute the GTP/GDP binding domain. In the crystal structure of EF-Tu, four peptide loops connecting beta sheets and alpha helices form the pocket for binding GDP. Studies using site-directed mutagenesis and immnochemical probes, indicate that the basic structure of the GDP binding site is conserved between p21 and EF-Tu. Furthermore, these studies also conclude that GTP binding is crucial for p21 ras cellular function. Although the precise target molecules for p21 are still unknown, the finding of the on/off switch function for ras genes have provided a better understanding of the mechanism of proto-oncogene activation, and may also provide further impetus to explore means of cancer intervention by interfering with the switch function.

Amino Acid Sequence↗

PubMed1985

Posttranslational processing of p21 ras proteins involves palmitylation of the C-terminal tetrapeptide containing cysteine-186.

The p21 proteins of ras oncogenes are synthesized as precursors in the cytosol. After processing, which involves acylation, the products are associated with the plasma membrane in eucaryotic cells. The p21 overproduced in Escherichia coli, however, is not processed by acylation. A synthetic tetrapeptide of the p21 C terminus is used to identify the acylation site in eucaryotic p21 as cysteine-186. The same peptide of bacterial p21 is not acylated. Although p21 of Harvey murine sarcoma virus-transformed NRK cells can be metabolically labeled with either [3H]palmitate or [3H]myristate, the lipid moiety of the hydrophobic peptide is identified as palmitic acid. We suggest that the enzymatic mechanism for p21 palmitylation may be different from N-terminal myristylation of many other membrane proteins.

Animals↗

PubMed1985

Biochemical properties of a highly purified v-rasH p21 protein overproduced in Escherichia coli and inhibition of its activities by a monoclonal antibody.

The v-rasH oncogene of Harvey murine sarcoma virus encodes a 21,000-dalton p21 protein which has been expressed at a high level as a fusion protein in Escherichia coli. We have purified the p21 to over 90% in purity without the use of any detergent or protein denaturant. The purified p21 possesses full biochemical activities of GTP/GDP binding, autokinase, and GTPase. Scatchard analysis indicates a single class of binding sites with Kd values of 0.83 X 10(-8)M for GTP and 1.0 X 10(-8)M for GDP. The binding site can be specifically labeled with a [3H]GTP photoaffinity analog, P3-(4-azidoanilido)-5' GTP. To probe for the active center of p21, we used a battery of six monoclonal antibodies to p21 to examine their effects on p21 activities. We found that only one monoclonal antibody, Y13-259, was capable of inhibiting both GTP/GDP binding and autokinase enzymatic activities, suggesting that these p21 activities are related activities conferred by a single active center within the p21 molecule. These observations together with the recent finding that microinjection of the same monoclonal antibody into NIH 3T3 cells specifically blocks p21 in vivo function (Mulcahy et al., Nature [London] 313:241, 1985) strongly suggest that p21 in vitro activities are responsible for its cellular function.

Animals↗

PubMed1984

Metabolic turnover of human c-rasH p21 protein of EJ bladder carcinoma and its normal cellular and viral homologs.

The EJ bladder carcinoma oncogene is activated by a point mutation in the c-rasH proto-oncogene at the 12th amino acid codon. In an attempt to understand the mechanism of oncogenic activation, a comparative study was undertaken to examine the metabolic turnover and subcellular localization of the p21 protein encoded by the EJ oncogene, the viral oncogene, and its normal cellular homolog. Pulse-labeling experiments indicated that both c-ras p21 proteins were synthesized by a very similar pathway, as was observed for the viral p21 protein of Harvey murine sarcoma virus. The pro-p21 proteins were detected in free cytosol, and the processed products were associated with plasma membrane. The intracellular half-life of p21 proteins was determined by pulse-labeling and chasing in the presence of excess unlabeled methionine. Although both p21 proteins of EJ and the normal c-ras genes which are not phosphorylated have a half-life of 20 h, the viral p21 protein of Harvey murine sarcoma virus which includes a phosphorylated form is much more stable in cells, having a half-life of 42 h, apparently due to phosphorylation.

Animals↗