Abstract
The kinase suppressor of Ras 1 (KSR1) has a fundamental role in mitogenic signaling by scaffolding components of the Ras/MAP kinase pathway. In response to Ras activation, KSR1 assembles a tripartite kinase complex that optimally transfers signals generated at the cell membrane to activate ERK. We describe a novel mechanism of ERK attenuation based on ubiquitin-dependent proteolysis of KSR1. Stimulation of membrane receptors by hormones or growth factors induced KSR1 polyubiquitination, which paralleled a decline of ERK1/2 signaling. We identified praja2 as the E3 ligase that ubiquitylates KSR1. We showed that praja2-dependent regulation of KSR1 is involved in the growth of cancer cells and in the maintenance of undifferentiated pluripotent state in mouse embryonic stem cells. The dynamic interplay between the ubiquitin system and the kinase scaffold of the Ras pathway shapes the activation profile of the mitogenic cascade. By controlling KSR1 levels, praja2 directly affects compartmentalized ERK activities, impacting on physiological events required for cell proliferation and maintenance of embryonic stem cell pluripotency.
Highlights
The ubiquitin–proteasome system (UPS) emerged as an important posttranslational mechanism that controls cell growth, differentiation, metabolism and survival
To map the praja[2] interacting motif, overlapping 25-mer peptides derived from human kinase suppressor of Ras 1 (KSR1) sequence were spotted onto a membrane and overlaid with purified glutathione S-transferase polypeptide (GST)-praja[21–531] fusion protein, as previously described.[32]
In vitro ubiquitination assays demonstrated that praja[2], but not its inactive mutant, directly ubiquitylates KSR1 (Figure 2f). These findings demonstrate that KSR1 is targeted by the ubiquitin pathway in response to stimulation of receptor tyrosine kinase (RTK) or G protein-coupled receptor (GPCR)
Summary
The ubiquitin–proteasome system (UPS) emerged as an important posttranslational mechanism that controls cell growth, differentiation, metabolism and survival. Praja[2] belongs to a growing family of widely expressed mammalian RING-H2 proteins with intrinsic E3 ubiquitin-ligase activity.[23,24,25,26] During GPCRcAMP stimulation, praja[2] ubiquitylates and degrades the inhibitory (R) subunits of PKA, sustaining downstream signals carried out by cAMP.[27] In proliferating cells, praja[2] promotes ubiquitindependent proteolysis of MOB1, a core component the tumor-suppressor Hippo cascade. Degradation of MOB1 through the UPS attenuates the Hippo cascade and sustains tumor growth.[28] A role of praja2UPS in neuronal differentiation and glucose homeostasis has been recently described.[29,30] the impact of praja[2] in the control of ERK signaling was unknown. By modulating KSR1ERK signaling, praja[2] profoundly impacts on essential aspects of embryonic stem cell (ESC) differentiation
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