The highly conserved kinase LIN-45, ortholog of human Braf, acts in the EGFR-Ras-Raf-MAPK signaling pathway and is essential for vulval induction in C. elegans. As Braf is frequently activated in human cancers, identification of conserved regulators that modulate LIN-45/Braf activity may provide insight into cancer biology. In our previous work, we found that LIN-45 protein is unstable in P6.p, the Vulval Precursor Cell (VPC) where EGFR signaling is highest. LIN-45 turnover is mediated by a cis-acting conserved Cdc4-phosphodegron (CPD) and requires the E3 ligase SEL-10/Fbw7, which targets phosphorylated CPD substrates for ubiquitination. LIN-45 turnover also requires MPK-1, the ERK ortholog downstream of LIN-45 in vulval induction, revealing a negative feedback loop in the Ras-Raf-MAPK cascade. MPK-1 likely acts to phosphorylate one moiety in the LIN-45 CPD; however, kinases responsible for additional sites are unknown. Because loss of sel-10 enhances LIN-45 activity in several cellular and genetic contexts, we expect that kinases required for LIN-45 turnover will similarly modulate Ras-Raf-MAPK activity.
To discover kinases that phosphorylate the LIN-45 CPD or otherwise regulate SEL-10 or Ras-Raf-MAPK activity, we created a visible GFP sensor for SEL-10/MPK-1-mediated LIN-45 degradation, and conducted an RNAi screen to identify kinases needed for its turnover. We will present new data indicating that LIN-45 protein turnover requires the conserved kinases GSK-3, CDK-4, and CDK-2, and that these kinases act cell-autonomously in VPCs. Furthermore, we find that loss of gsk-3, cdk-4, or cdk-2 enhances the Multivulva phenotype caused by a hyperactive form of LIN-45, confirming that these kinases negatively regulate Ras-Raf-MAPK activity. Since CDK-4 and CDK-2 are critical regulators of G1 and G1/S cell-cycle phase progression, we have examined how LIN-45 turnover is coordinated with cell cycle control. In wild-type, LIN-45 is expressed in all VPCs during L2 when these cells are quiescent. Upon entry to L3, when CDKs become active and VPCs resume cell cycle progression, LIN-45 is rapidly downregulated in P6.p. We find that precocious activation of CDK activity causes LIN-45 downregulation to occur prematurely in L2, suggesting that CDK activity normally restricts LIN-45 turnover to the L3 stage. Based on these results, we propose that the dynamic developmental pattern of LIN-45 protein stability in VPCs is a result of at least two inputs: 1) spatial information provided by MPK-1 activity in P6.p, and 2) temporal information mediated by the reactivation of CDK activity in the L3 stage.
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