Worm Breeder's Gazette 16(3): 29 (June 1, 2000)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Activation of the ras-MAP kinase pathway promotes protein degradation in muscle

Nate Szewczyk, Lew Jacobson

Dept. of Biological Sciences, Univ. of Pittsburgh, Pittsburgh PA 15260

      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.

(Many thanks to Dave Eisenmann and the CGC for strains. Supported by NSF MCB-9630841.)

[1] Zdinak et al., J. Cell. Bioch. 67: 143-153 (1997)
[2] Szewczyk et al., J. Cell. Sci., in press (2000)
[3] Kokel et al., Genes & Development 12: 1425-1437 (1998)
[4] Selfors et al., PNAS 95: 6903-6908 (1998)
[5] Sieburth et al., Cell 94: 119-130 (1998)
[6] Li et al., Genes Develop. 14:895-900 (2000)