Biomedical subjects

I Chackalaparampil

Publications and source records attributed to I Chackalaparampil.

5 recordsLinked to original sources

PubMed1992

Role of p34cdc2-mediated phosphorylations in two-step activation of pp60c-src during mitosis.

Phosphorylation of pp60c-src by p34cdc2 at three amino-proximal serine/threonine residues is temporally correlated with, but insufficient for, mitotic activation of c-Src kinase. The direct cause of activation during mitosis appears to be temporally correlated partial dephosphorylation of Tyr-527, a residue whose phosphorylation strongly suppresses pp60c-src activity. Site-directed mutagenesis of the serine/threonine phosphorylation sites blocks half the mitosis-specific decrease in Tyr-527 phosphorylation and half the increase in pp60c-src kinase activity. We conclude that p34cdc2 partially activates pp60c-src by a two-step process in which its serine/threonine phosphorylations either sensitize pp60c-src to a Tyr-527 phosphatase or desensitize it to a Tyr-527 kinase. Furthermore, additional events, independent of these p34cdc2-mediated phosphorylations, participate in mitotic activation of pp60c-src.

3T3 Cells↗

PubMed1992

c-Src and mitosis.

The transforming potential and by inference the physiological function of the proto-oncoprotein pp60c-src closely correlate with the level of its protein tyrosine kinase activity. We have investigated the cell cycle-dependent regulation of this activity using mouse fibroblasts overexpressing chicken or mouse pp60c-src as a model system. During mitosis pp60c-src becomes phosphorylated at specific serine and threonine residues by p34cdc2. At the same time its tyrosine kinase activity, assayed in vitro, is increased approximately twofold and accessibility of its SH2 domain for binding relevant phosphotyrosine-containing ligands increases by about 15-fold. A kinase-defective mutant of pp60c-src exhibits a substantial (50-70%) decrease in phosphorylation at Tyr527 during mitosis. Phosphorylation of this residue negatively regulates kinase activity. Indirect evidence indicates a lesser decrease in wild-type pp60c-src Tyr527 phosphorylation during mitosis. Coordinate mutation of the mitosis-specific phosphorylation (MSP) sites in kinase-defective pp60c-src greatly reduces, though does not abolish, its mitosis-specific tyrosine dephosphorylation. Similarly, coordinate mutation of the three MSP sites in chicken pp60c-src or the corresponding two sites in mouse pp60c-src does not completely block mitotic stimulation of kinase activity. Thus, additional events beyond p34cdc2-mediated phosphorylation are involved in cell-cycle dependent regulation of pp60c-src activity. This is also suggested by the stimulation of pp60c-src kinase activity and decrease in phosphorylation of Tyr527 observed following treatment of fibroblasts with okadaic acid, a potent inhibitor of types 1 and 2A serine/threonine phosphatases. The potential role of cell cycle-dependent regulation of phosphatases and kinases acting on the regulatory tyrosine residue of pp60c-src is discussed.

Animals↗

PubMed1991

Altered tyrosine 527 phosphorylation and mitotic activation of p60c-src.

The tyrosine kinasee activity of p60c-src, the protein product of the c-src gene, increases during mitosis; this may be important in initiating at least some of the cellular changes that occur during this phase of the cell cycle. Although there is evidence that p60c-src is phosphorylated at several sites during mitosis, phosphorylation in vitro does not increase its kinase activity. We now report that the kinase activity of a p60c-src mutant with residue tyrosine 527 changed to phenylanine does not change during the cell cycle, suggesting that changes in the phosphorylation state of this residue may be responsible for the activation of p60c-src at mitosis. Although changes in phosphorylation at Tyr 527 cannot be detected with the wild-type protein we find that phosphorylation at Tyr 527 of a mutant with reduced kinase activity decreases threefold during mitosis. On the basis of these results we suggest that activation of p60c-src at mitosis results from decreased phosphorylation on Tyr 527, and that p60c-src may be or may activate the kinase that phosphorylates Tyr 527.

Animals↗

PubMed1988

Altered phosphorylation and activation of pp60c-src during fibroblast mitosis.

At least half the pp60c-src in NIH 3T3-derived c-src overexpresser cells in modified by novel threonine and, possibly, serine phosphorylation within its amino 16 kd region during mitosis. At the same time, the tryptic phosphopeptide containing Ser 17, the site of cyclic AMP-dependent phosphorylation, is either modified or dephosphorylated. While the amount of pp60c-src is not significantly altered, the in vitro-specific kinase activity of modified pp60c-src is enhanced 4- to 7-fold. Modified pp60c-src has the same tyrosine-containing tryptic phosphopeptides as pp60c-src from unsynchronized cells, indicating that activation is independent of Tyr 416/Tyr 527 phosphorylation. Electrophoretic mobility retardations indicated that endogenous pp60c-src and pp60v-src are similarly modified during mitosis. The modifications and enhanced activity disappear near the time of cell division. These results suggest that pp60c-src is regulated by and, in turn, may regulate mitosis-specific events in fibroblasts.

Animals↗

PubMed1985

Altered processing of a major secreted phosphoprotein correlates with tumorigenicity in Rous sarcoma virus-transformed mammalian cells.

Anchorage-independent growth is highly correlated with neoplastic growth in vivo, and the retinoids (vitamin A and its analogs) inhibit this property in a wide variety of oncogenically transformed cells. We report here that retinoic acid-treated Rous sarcoma virus-transformed rat (RR1022) and vole (SR-1T) cells, which show reversible loss of anchorage-independent growth and assume nontransformed morphology, secrete a major 69-kilodalton phosphoprotein (pp69) instead of the 62-kilodalton phosphoprotein (pp62) secreted by their untreated counterparts. As determined by V8 protease mapping and by two-dimensional electrophoretic analysis, this 69-kilodalton polypeptide was indistinguishable from the pp69 released by nontransformed normal rat kidney cells. Neither retinoic acid-treated RR1022 cells nor normal rat kidney cells secreted pp62, and retinoic acid treatment did not have any significant effect on the synthesis, subcellular localization, or phosphokinase activity of pp60src. Furthermore, treatment with retinoic acid did not alter the synthesis of the transformation-specific 53-kilodalton phosphoprotein (p53) and secretion of the transforming growth factors in RR1022 cells. Our studies showed that there is a clear correlation between the release of pp69 or pp62 and the ability of cells to grow in vitro with or without anchorage. This may provide an important clue for elucidating specific biochemical events involved in anchorage regulation of growth.

Animals↗