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Reversal of Raf-1 activation by purified and membrane-associated protein phosphatases
P Dent1, T Jelinek, D K Morrison
1Howard Hughes Medical Institute, University of Virginia, Charlottesville 22908, USA.
Abstract:
The Raf-1 protein kinase participates in transduction of mitogenic signals, but its mechanisms of activation are incompletely understood. Treatment of human Raf-1 purified from insect Sf9 cells co-expressing c-H-Ras and Src(Y527F) (in which phenylalanine replaces tyrosine at residue 527) with either serine-threonine or tyrosine phosphatases resulted in enzymatic inactivation of Raf-1. Inactivation of purified Raf-1 was blocked by addition of either the 14-3-3 zeta protein or heat shock protein 90. Loading of plasma membranes from transformed cells with guanosine triphosphate (GTP) resulted in inactivation of endogenous or exogenous Raf-1; inactivation was blocked by inclusion of protein phosphatase inhibitors. These results suggest the existence of protein phosphatases in the cell membrane that are regulated by GTP and are responsible for Raf-1 inactivation.
Insights
Protein phosphatases in cell membranes inactivate Raf-1 kinase activity. This inactivation is regulated by guanosine triphosphate (GTP) and can be blocked by specific proteins and inhibitors, suggesting a novel regulatory mechanism for mitogenic signaling.
Area of Science:
- Cellular signaling pathways
- Protein kinase regulation
- Molecular mechanisms of signal transduction
Background:
- Raf-1 protein kinase is crucial for transmitting signals that promote cell growth (mitogenic signals).
- The precise mechanisms by which Raf-1 is activated remain unclear.
- Understanding Raf-1 regulation is key to deciphering cellular growth control.
Purpose of the Study:
- To investigate the mechanisms of Raf-1 inactivation.
- To identify factors involved in regulating Raf-1 activity.
- To explore the role of phosphatases in Raf-1 regulation.
Main Methods:
- Purified human Raf-1 was treated with serine-threonine and tyrosine phosphatases.
- Enzymatic activity of Raf-1 was assessed after phosphatase treatment.
- Plasma membranes from transformed cells were loaded with guanosine triphosphate (GTP).
- The effect of 14-3-3 zeta protein and heat shock protein 90 on Raf-1 inactivation was examined.
Main Results:
- Treatment with serine-threonine or tyrosine phosphatases inactivated purified Raf-1.
- Inactivation of purified Raf-1 was prevented by 14-3-3 zeta protein and heat shock protein 90.
- Loading plasma membranes with GTP led to inactivation of endogenous and exogenous Raf-1.
- Protein phosphatase inhibitors blocked GTP-induced Raf-1 inactivation.
Conclusions:
- Cell membrane-associated protein phosphatases, regulated by GTP, are responsible for Raf-1 inactivation.
- These findings reveal a novel regulatory pathway for Raf-1 kinase activity.
- This discovery has implications for understanding mitogenic signal transduction and cellular proliferation control.