Presentation/Session Information

Session Information

Session Title: Cell Fate, Differentiation and Morphogenesis Session Type: Parallel
Session Location: Northwest Auditorium Session Time: Sat, Jun 27 8:30AM - 11:30AM

Presentation Information

Program Number: 182 Presentation Time: 9:30AM - 9:42AM

Presentation Content

The conserved kinases MPK-1, GSK-3, CDK-4 and CDK-2 promote LIN-45/Braf protein turnover in a dynamic spatial and temporal pattern.Claire de la Cova, Iva Greenwald. HHMI and Dept Biochem, Columbia Univ, New York,

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.

Please note: Abstract shown here 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.

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