Negative feedback by conserved kinases patterns degradation of C. elegans Raf in vulval fate patterning

Abstract

Activation of a canonical EGFR-Ras-Raf-ERK cascade initiates patterning of multipotent Vulval Precursor Cells (VPCs) of C. elegans. We previously showed that this pathway includes a negative-feedback component in which MPK-1/ERK activity targets the upstream kinase LIN-45/Raf for degradation by the SEL-10/FBXW7 E3 ubiquitin ligase. This regulation requires a Cdc4 phosphodegron (CPD) in LIN-45 that is conserved in BRAF. Here, we identify and characterize the minimal degron that encompasses the CPD and is sufficient for SEL-10-mediated, MPK-1-dependent protein degradation. A targeted screen of conserved protein kinase-encoding genes yielded gsk-3/GSK3 and cdk-2/CDK2 as required for LIN-45 degron-mediated turnover. Genetic analysis revealed that LIN-45 degradation is blocked at the L2 stage due to cell cycle quiescence, and that relief of the block during the L3 stage relies on activation of CDKs. Additionally, activation of MPK-1 provides spatial pattern to LIN-45 degradation but does not bypass the requirement for gsk-3 and cdk-2 activity. This analysis supports a model whereby mpk-1/ERK, gsk-3/GSK3, and cdk-2/CDK2, along with sel-10/FBXW7, constitute a regulatory network that exerts spatial and temporal control of LIN-45/Raf degradation during VPC patterning.