Abstract
Previously pp60v-src, cyclin A, p39mos, and maturation-promoting factor (composed of Cdc2 and cyclin B) have been shown to activate mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK) in cell-free extracts of Xenopus oocytes. The pp60v-src pathway is dependent on a functional Ras signal whereas the cyclin/maturation-promoting factor pathway is not. Here we show that protein kinase C (PKC) is also able to stimulate MAPK in a Ras-dependent manner, but PKC is not necessary for signaling by pp60v-src. In addition, preincubation of extracts with cAMP-dependent protein kinase (PKA) blocks stimulation of MAPK by cyclin, p21V12ras, PKC, or pp60v-src, by at least 50%, but stimulation by c-Mos is unaffected. Furthermore, inhibition of endogenous PKA by the heat-stable PKA inhibitor is sufficient to stimulate MAPK activity in these extracts in the absence of protein synthesis and without dependence on a functional Ras protein. These results suggest that independent pp60v-src and PKC pathways converge at Ras and that PKA acts to block MAPK activation by both Ras-dependent and -independent signals.
Highlights
Mitogen-activated proteinkinases (MAPKs)' are activated in response to a number of signal transduction pathways,including those stimulated by tyrosine kinases [1,2,3,4,5], protein kinase C (PKC, 6, 71, and progesterone in Xenopus oocytes [8,9,10,11]
MAP& are phosphorylated and activated byMAPK kinases (MEKs), which can phosphorylate both the tyrosine and threonine residues inMAPK required for activation [12,13,14,15,16,17,18,19]
MEKs themselves are activated by phosphorylation on serine/ threonine residues by MAPK kinase kinases (MEKKs, 13, 18, 20)
Summary
Are able to activateMAI'K in response to addition ofpp60'", Female Xenopus laevis were obtained from Xenopus I (Ann Arbor, p21Ha-ras, ~ 3 9 ~ - ~a'n' ,d cyclidCdc ( 5 , 72-74). Purification of baculovirus-expressed pp60""" was carried oubt y immunoaffintation (analogous to Leu'' in mammalianHa-Ras) was added t o extracts prior to PKC-M addition. This cytoplasmically restricted p21 protein, designated RAST,binds GTPase-activating protein with much greater affinity than c-Ras and exertsa dominant-negative effect on signa1in.gthrough p21 ( 5 , 69). To. S-transferase-c-Mos fusion construct was prepared, expressed in Sf9 investigatethis possibility, extracts were pretreated witha cells, and purified as described elsewhere.' The EGF receptor peptide PKC pseudosubstrate inhibitor peptide(PKC-I) that is well (EGFR,,cs6,, RRELVEPLTPSGEAPNQALLR) for use as a MAPK substrate [70] was synthesized by a local peptide synthesis facility.
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