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Mitogen-induced tyrosine phosphorylation of 41 kDa and 43 kDa proteins. Potential role in integrating multiple
Abstract:
We have examined the possible involvement of pertussis toxin (PT)-sensitive GTP-binding protein and protein kinase C (PKC) in mitogen-induced tyrosine phosphorylation of the 41 kDa and 43 kDa cytosol proteins using PT-pretreated (inactivation of PT-sensitive GTP-binding protein) or phorbol 12-myristate 13-acetate (PMA)-pretreated (depletion of PKC) mouse fibroblasts. The effects of the inactivation of PT-sensitive GTP-binding protein and the depletion of PKC on mitogen-stimulated tyrosine phosphorylation of the proteins were similar and varied significantly and systematically in response to growth factors. The important finding was that such inhibitory effects of PT-sensitive GTP-binding protein inactivation and PKC depletion on protein tyrosine phosphorylation induced by each mitogen always correlated well with their inhibitory effects on each mitogen-stimulated DNA synthesis. Although the extent of platelet-derived-growth-factor-induced phosphorylation of the proteins was decreased to approx. 50% in PT- and PMA-pretreated cells compared with native cells, protein phosphorylation itself was not affected and occurred at identical sites on each protein in native, PT- and PMA-pretreated cells. These results suggest that: (1) 41 kDa and 43 kDa proteins are located downstream of PT-sensitive GTP-binding protein and PKC in the mitogenic signalling pathways of growth factors, (2) protein phosphorylation occurs via a cascade of events which includes the activation of the receptor tyrosine kinases, PKC and other unidentified kinase(s) which directly participate(s) in the phosphorylation of the 41 kDa and 43 kDa proteins, and (3) their phosphorylation may play an important role in integrating multiple mitogenic signalling pathways.
Insights
Pertussis toxin (PT)-sensitive GTP-binding protein and protein kinase C (PKC) are crucial for mitogen-induced tyrosine phosphorylation of cytosol proteins, impacting DNA synthesis in mouse fibroblasts.
Area of Science:
- Cellular signaling pathways
- Molecular biology
- Biochemistry
Background:
- Mitogen-induced cell proliferation involves complex signaling cascades.
- Tyrosine phosphorylation is a key regulatory mechanism in cell growth.
- The roles of specific signaling molecules like GTP-binding proteins and protein kinase C in these processes require elucidation.
Purpose of the Study:
- To investigate the involvement of pertussis toxin (PT)-sensitive GTP-binding protein and protein kinase C (PKC) in mitogen-induced tyrosine phosphorylation.
- To determine the relationship between this phosphorylation and mitogen-stimulated DNA synthesis.
Main Methods:
- Mouse fibroblasts were pretreated with PT to inactivate PT-sensitive GTP-binding protein.
- Fibroblasts were pretreated with phorbol 12-myristate 13-acetate (PMA) to deplete PKC.
- Tyrosine phosphorylation of 41 kDa and 43 kDa cytosol proteins was analyzed in response to various mitogens.
- DNA synthesis was measured to assess cellular proliferation.
Main Results:
- Inactivation of PT-sensitive GTP-binding protein and depletion of PKC similarly affected mitogen-stimulated tyrosine phosphorylation of 41 kDa and 43 kDa proteins.
- These effects correlated with inhibitory effects on mitogen-stimulated DNA synthesis.
- Platelet-derived growth factor-induced phosphorylation was reduced by approximately 50% in pretreated cells, but phosphorylation sites remained unchanged.
Conclusions:
- PT-sensitive GTP-binding protein and PKC are located downstream of growth factor signaling pathways.
- Protein phosphorylation involves a cascade including receptor tyrosine kinases, PKC, and other kinases.
- Phosphorylation of 41 kDa and 43 kDa proteins may integrate multiple mitogenic signaling pathways.