We are studying the regulation of protein degradation, using transgenic strains of C. elegans expressing a soluble, enzymatically active
unc-54::lacZ fusion protein in body-wall and vulval muscles. This fusion protein is completely stable in well-fed animals, but is degraded upon starvation by activation of a pre-existing proteolytic system [1]. Degradation can be prevented by stimulating the nicotinic acetylcholine receptor (nAChR) [2] or intracellular calcium release or by activating protein kinase C (PKC) or by inhibitors of proteasome activity. Because ras signaling is known to interact with calcium and PKC signaling, we constructed strains containing various
let-60 ras mutant alleles and the
unc-54::lacZ reporter. Well-fed animals homozygous for the temperature-activated ras allele
ga89 express wild-type levels of fusion protein (by X-gal staining, by fluorometric activity assay and by Westerns) when grown at 16C, but when shifted to 25C as adults show a time-dependent loss of lacZ activity and of reporter protein. This does not happen in similarly treated wild type animals, and is less pronounced in constitutively-activated
let-60 (
n1046) mutants. Degradation also occurs (in a smaller fraction of individuals) in strains homozygous for
gap-1 (
ga133) or
gap-2 (
pe103) mutations that increase ras activation, and the
gap-1 (
ga133) mutation enhances the rate of protein degradation at 25C in a strain carrying
let-60 (
ga89). These data support the hypothesis that protein catabolism is stimulated by ras activation rather than by some other peculiar property of
ga89 mutant ras protein. Cycloheximide treatment from the time of temperature shift does not prevent protein breakdown, implying that ras-induced protein catabolism does not depend upon ras-induced gene expression, but rather uses pre-existing signaling pathways and proteases. Because
let-60 (
ga89) produces a "clear" (Clr) phenotype but
n1046 does not, we also examined the ability of
clr-1 (
e1745) to induce muscle protein degradation. (
clr-1 encodes a protein tyrosine phosphatase [3].) Animals homozygous for this ts loss-of-function allele catabolize the reporter at 25C but not at 16C; this degradation, like the Clr phenotype, is suppressed by a loss of function mutation in
egl-15 (
n1783), which encodes a fibroblast growth factor receptor homologue. It is possible but by no means certain that the
clr-1 loss-of-function mutation stimulates protein catabolism by activating the ras pathway. Mutations in
soc-2/sur-8 suppress
clr-1 loss-of-function [4] and suppress activated
let-60 [5]. The
soc-2 gene (aka
sur-8) encodes a "leucine rich repeat" protein [4,5] that has been reported to promote ras binding to the protein kinase raf [6]. We find that
clr-1-induced protein catabolism is suppressed in a
clr-1 (
e1745);
soc-2 (
n1774) strain and are currently determining if a
soc-2/sur-8 mutation suppresses protein degradation in
let-60 (
ga89)
soc-2 (
n1774) animals. Suppression by
soc-2/sur-8 suggests that the output to proteolysis from activated
let-60 ras might be transduced by the raf-MEK-MAP kinase pathway. We find that the effect of activated ras is suppressed by
mpk-1 (
n2521), indicating that downstream signal is transduced by MPK-1 MAP kinase. The Clr phenotype of
ga89 is also suppressed by
mpk-1 (
n2521). Although the known signal outputs from MAP kinase are primarily in the nucleus, this represents a case of non-transcriptional signalling by the ras-MAP-kinase pathway.