A dominant-negative mutant of raf blocks mitogen-activated protein kinase activation by growth factors and oncogenic p21ras.

D Schaap,J Van Der Wal,L.R Howe,C.J Marshall,W.J Van Blitterswijk

doi:10.1016/s0021-9258(20)80719-4

D Schaap, J Van Der Wal + Show 3 more

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https://doi.org/10.1016/s0021-9258(20)80719-4

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Abstract

p74raf-1, a serine/threonine kinase, is structurally related to the protein kinase C (PKC) family and contains a cysteine motif in its N-terminal domain, which is essential for its regulation. It has been shown that p74raf-1 functions upstream of mitogen-activated protein (MAP) kinase kinase. We have constructed a p74raf-1 mutant (N delta raf) that only contains the N-terminal regulatory domain. When transiently expressed in COS-M6 cells, N delta raf efficiently blocked the activation of the MAP extracellular signal regulated kinase (ERK2), induced by either epidermal growth factor, phorbol ester, serum, or oncogenic p21ras. Similar constructs with the cysteine motifs from either PKC-alpha or diacylglycerol kinase did not inhibit activation of ERK2. Overexpression of full-length p74raf-1 rescued the inhibition of ERK2 by N delta raf in a stimulus dependent manner, indicating that N delta raf acts as a competitive inhibitor of wild-type p74raf-1. In contrast, overexpression of either PKC-alpha, -epsilon, or -zeta in N delta raf-containing cells could not rescue the inhibition of ERK2. We conclude that p74raf-1 is an essential mediator of epidermal growth factor- and phorbol ester-induced ERK2 activation and that the MAP kinase kinase activity of p74raf-1 cannot be substituted with either PKC-alpha, -epsilon or -zeta.

Highlights

From the Divisionof Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CXAmsterdam, The Netherlands and the $Institute of Cancer Research, Chester BeattyLaboratories, Fulham Road, London SW3 6JB, United Kingdom p74"P1, a serindthreonine kinase, is structurally re- be the substrate(diacylglycerol) binding site( 5 ) .It is unknown lated to the protein kinase C (PKC) family and contains what molecule(s) may bind tothesingle cysteine motif of a cysteine motif in its N-terminal domain, which is es- p74"fi1, an unidentifiedactivatordownstream of sential for its regulation
We conclude thatp74"f-I is an essential mediator of epidermal growth factor- and phorbol ester-induced ERK2 activation and thatthe MAP kinase kinase kinase activity ofp74-f-I cannotbesubstituted with either
NAraf efficiently blocked the activation of ERK2 tag in response to EGF, TPA, and serum stimulation,as shown by the absence of a shifted form on SDS-gel (Fig. 1B).Slower migrationof ERK2 on SDS-gelhas previously been reported to correlate wpihthosphorylation and activation of ERK2

Summary

Introduction

From the Divisionof Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CXAmsterdam, The Netherlands and the $Institute of Cancer Research, Chester BeattyLaboratories, Fulham Road, London SW3 6JB, United Kingdom p74"P1, a serindthreonine kinase, is structurally re- be the substrate(diacylglycerol) binding site( 5 ) .It is unknown lated to the protein kinase C (PKC) family and contains what molecule(s) may bind tothesingle cysteine motif of a cysteine motif in its N-terminal domain, which is es- p74"fi1, an unidentifiedactivatordownstream of sential for its regulation. When transientlyexpressed in COS-MI cells, ~ 7 4 ' ~ fi-sIactivated by various growthfactors, phorbol esters, NAruf efficiently blocked the activation of the MAP ex- and oncogenic ~21'"". NAraf is similarto a previously described mutant, raf-C4 (61, but contains a 12amino acid longC-terminalepitope tag from DG kinase (PPRSTNFFGFLS)which is recognized by antiserum 13572 (Fig. IA).To determine if NAraf inhibits wild-type ~74'~f-'w, e tested whether NAraf blocked ERK2 activation.

Results

Conclusion