Worm Breeder's Gazette 17(2): 28 (April 1, 2002)

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.

A DAF-2 pathway regulates muscle protein degradation by antagonizing Ras-Raf-MEK-MAPK signaling

Nate Szewczyk, Brant Peterson, Sami Barmada, Leah Parkinson, Lew Jacobson

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

We have been using the non-myofibrillar myosin::ß-galactosidase fusion protein produced by ccIs55(unc-54::lacZ) to "report" on protein degradation in muscle. This protein is stable in well-fed wild-type animals, but its degradation is induced by starvation, by acute disruption of cholinergic signaling (e.g., cha-1(p1182ts) animals) [1], or by activation of the Ras-MAPK pathway either directly (e.g., let-60(ga89ts) [2]) or indirectly (clr-1(e1745ts) activation of EGL-15 FGFR). Protein degradation induced by starvation or denervation is distinct from Ras-induced degradation on three grounds: (a) Reduction-of-function mutations in mek-2 (MEK) or mpk-1 (MAPK) suppress Ras-induced but not starvation/denervation-induced degradation; (b) Proteasome inhibitors block starvation/denervation-induced but not Ras-induced degradation; (c) The nAChR agonist levamisole blocks starvation/denervation-induced but not Ras-induced degradation.

Because of the well-known effects of insulin and Insulin-like Growth Factor (IGF) in promoting protein anabolism in mammalian muscle, we have now explored a possible role in muscle for the signaling pathway downstream of the DAF-2 IGFR homologue. We find that well-fed daf-2(e1370ts) or (m41ts) animals degrade the pre-existing reporter protein when shifted to nonpermissive temperature. Specifically, animals grown until mid-adulthood at 16°C and then shifted to 25°C showed a time-dependent loss of reporter activity and protein. Similarly, reporter degradation was observed when age-1(hx546ts) mutants were shifted to 25°C or when wild-type animals were treated with the PI-3-kinase (AGE-1) inhibitor LY-290042 [3]. This protein degradation appears to utilize pre-existing signaling cascades and proteases, inasmuch as degradation can still be triggered after treatment with the protein synthesis inhibitor cycloheximide.

The expression of age-1+ in muscle is known to affect lipid storage [4]. We infer that AGE-1 affects muscle protein degradation by intra-muscular action, because expression of age-1+ from the unc-54 promoter (constructs kindly provided by C. Wolkow & G. Ruvkun) is sufficient to block protein degradation in the muscles of age-1 null-mutant animals. Protein degradation induced by daf-2(m41ts) or LY-294002 is also prevented when the downstream signaling pathway is activated by either (a) a daf-18 reduction-of-function mutation that permits accumulation of the AGE-1 product PtdIns-P3; or (b) gain-of-function mutations pdk-1(mg142) or akt-1(mg144). However, the reduction-of-function mutation pdk-1(sa709) is not sufficient to trigger protein degradation, suggesting either that PDK-1 can activate AKT-1 but is not required for AKT-1 activation, or that sa709 mutants retain sufficient PDK-1 function to activate AKT-1. Thus, IGFR-PI3K-(PDK)-Akt signals oppose protein degradation in muscle. In contrast to the way this pathway functions in controlling dauer larva formation, here the target of AKT-1 is not the forkhead-class transcription factor DAF-16, since degradation in LY-294002-treated animals is not blocked by the loss-of-function mutation daf-16(mgDf50).

What is the relevant target of AKT-1 action in muscle? Inhibitory phosphorylation of mammalian Raf by Akt has been reported [5], the mutation of presumptive Akt phosphorylation sites in LIN-45 Raf affects vulval development [6], and we had noted a low-frequency MuV phenotype in daf-2(m41) animals. These observations prompted us to test if the Ras-Raf-MEK-MAPK pathway was the target of negative regulation by the IGFR-PI3K-Akt pathway in worm muscle. Indeed, we found that reduction-of-function mutations soc-2(n1774), lin-45(sy96), mek-2(ku114) or mpk-1(n2521) suppressed protein degradation in response to LY-290042 treatment, but let-60(n2021) did not. Conversely, increased activity of the DAF-2 pathway is sufficient to block protein degradation induced by EGL-15 activation (clr-1(e1745ts); daf-18(e1375) animals). Thus, our results suggest that DAF-2 signals via AGE-1, PDK-1 and AKT-1 to inhibit LIN-45 Raf and protein degradation in muscle. They also imply that in normal adult muscle there is incoming signal to Raf, passed to subsequent steps in the cascade only when the balancing Akt inhibition of Raf is released.

Does the DAF-2 pathway affect the response to starvation? We find that starvation-induced reporter protein degradation is normal in daf-2(e1370ts) animals grown at 20°C then shifted to 25°C at the onset of starvation. However, the same mutants grown from L3 on at 25°C are at least partially resistant to reporter degradation in response to starvation at 25°C. These observations appear to reflect the excess lipid storage in daf-2 mutants at 25°C. We have noted in wild-type that the onset of protein degradation about 8 hr. after starvation corresponds approximately to the time required to exhaust stored lipid in the intestine (by Sudan Black staining). Furthermore, we observed normal starvation-induced protein degradation in age-1(hx546ts) animals grown at 20°C to adulthood then starved at 25°C, and in wild-type animals treated with LY-290042 from the time of starvation. We infer that DAF-2 signaling does not directly regulate muscle proteolysis in response to starvation.

[1] Szewczyk, Hartman, Barmada & Jacobson, J. Cell Sci. 113:2003-2010 (2000)
[2] Szewczyk, Peterson & Jacobson, Mol. Cell. Biol. (in press)
[3] Babar, Adamson, Walker, Walker & Lithgow, Neurobiol. Aging 20:513-519 (1999)
[4] Wolkow, Kimura, Lee & Ruvkun, Science 290:147-150 (2000)
[5] Zimmermann & Moelling, Science 286:1741-1744 (1999)
[6] Chong, Lee & Guan, EMBO J. 20:3716-3727 (2001)