Activation of Ras and the Mitogen-Activated Protein Kinase Pathway Promotes Protein Degradation in Muscle Cells of Caenorhabditis elegans

Abstract

To discover and study intracellular signals that regulate proteolysis in muscle, we have employed transgenic strains of Caenorhabditis elegans that produce a soluble LacZ reporter protein limited to body-wall and vulval muscles. This reporter protein is stable in well-fed wild-type animals, but its degradation is triggered upon a shift to 25°C in a strain carrying a temperature-sensitive activating mutation in the Ras oncogene homologue let-60. These mutants are not physiologically starved, inasmuch as growth rates are normal at 25°C. Rasinduced degradation is not prevented by the presence of cycloheximide added at or before the temperature shift and thus uses preexisting proteolytic systems and signaling components. Furthermore, degradation is triggered when adult animals are shifted to conditions of 25°C, confirming that Ras acutely promotes protein degradation in muscles whose developmental history is normal. Reduction-of-function mutations in the downstream protein kinase Raf (lin-45), MEK (mek-2), or mitogen-activated protein kinase (MAPK) (mpk-1) prevent Ras-induced protein degradation, whereas activated MPK-1 is sufficient to trigger degradation, indicating that this kinase cascade is the principal route by which Ras signaling triggers protein degradation in muscle. This pathway is activated in hypodermal cells by the LET-23 epidermal growth factor receptor homologue, but an activating mutation in let-23 does not promote proteolysis in muscle. Starvation-induced LacZ reporter degradation is unaffected by reduction-of-function mutations in Ras, Raf, MEK, or MAPK, implying that Ras activation and starvation trigger proteolysis by mechanisms that are at least partially independent. This is the first evidence that Ras-Raf-MEK-MAPK signaling activates protein degradation in differentiated muscle.The regulation of proteolysis in muscle is a clinically significant problem because of its relevance to muscle wasting and atrophy in skeletal muscle and pathological hypertrophy and atrophy in cardiac muscle. Muscle proteolysis is promoted by fasting or chronic starvation (18, 29), by disuse or denervation (19,31), in sepsis (10), and in cancer cachexia (53). Catastrophic loss of muscle protein is associated with increased mortality in patients with cancer, diabetes, sepsis, heart failure, or AIDS (30, 52), and extreme muscle wasting can itself be the proximal cause of death.The molecular mechanisms of proteolysis in muscle have been extensively studied (3, 4, 15, 31-33, 44, 45, 51), although there is considerable uncertainty remaining about the specific roles of the proteolytic systems involved. Moreover, several external signals that regulate muscle proteolysis have been identified but we know little about how intramuscular signaltransduction mechanisms might couple external stimuli to the regulation of protein degradation.We have been developing a genetically tractable model for studying the regulation of proteolysis in muscle, using strains of the nematode Caenorhabditis elegans that express a transgenecoded LacZ ...

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