Cdc37 Regulates Ryk Signaling by Stabilizing the Cleaved Ryk Intracellular Domain

Jungmook Lyu,Robin L Wesselschmidt,Wange Lu

doi:10.1074/jbc.m900207200

Abstract

Ryk is a Wnt receptor that plays an important role in neurogenesis, neurite outgrowth, and axon guidance. We have reported that the Ryk receptor is cleaved by gamma-secretase and that its intracellular domain (ICD) translocates to the nucleus upon Wnt stimulation. Cleavage of Ryk and its ICD is important for the function of Ryk in neurogenesis. However, the question of how the Ryk ICD is stabilized and translocated into the nucleus remains unanswered. Here, we show that the Ryk ICD undergoes ubiquitination and proteasomal degradation. We have identified Cdc37, a subunit of the molecular chaperone Hsp90 complex, as a Ryk ICD-interacting protein that inhibits proteasomal degradation of the Ryk ICD. Overexpression of Cdc37 increases Ryk ICD levels and promotes its nuclear localization, whereas Cdc37 knockdown reduces Ryk ICD stability. Furthermore, we have discovered that the Cdc37-Ryk ICD complex is disrupted during neural differentiation of embryonic stem cells, resulting in Ryk ICD degradation. These results suggest that Cdc37 plays an essential role in regulating Ryk ICD stability and therefore in Ryk-mediated signal transduction.

Highlights

Wnt signaling plays an essential role in several developmental processes, including cell proliferation, cell migration, and cell fate determination [1,2,3]
We show that the Ryk intracellular domain (ICD) is degraded following disruption of the Cdc37-Ryk ICD complex during the course of neural differentiation of embryonic stem cells (ESCs)
To prepare RykϪ/Ϫ ESCs, blastocysts were flushed from the uteruses of pregnant females 3.5 days after mating a Rykϩ/Ϫ breeder pair, and cells were explanted on feeder layers of irradiated mouse embryonic fibroblasts in growth medium containing 80% KnockoutTM Dulbecco’s modified Eagle’s medium (Invitrogen), 15% KnockoutTM serum replacement (Invitrogen), 5% fetal bovine serum (ESC-qualified, HyClone), 2 mM L-glutamine, 1ϫ nonessential amino acids, 1ϫ penicillin/streptomycin (Invitrogen), 1000 units/ml mouse leukemia inhibitory factor (LIF; Millipore), and 0.1 mM 2-mercaptoethanol (Invitrogen)

Summary

Present address

A comparison of Ryk intracellular domains from several species with catalytically active RTKs shows amino acid substitutions in subdomains I and II, suggesting a loss of function at the ATP binding site. In the absence of Notch binding, the cleaved ICD of the Notch ligand Delta is degraded by the proteasomal machinery [18] All of these observations suggest that the Ryk ICD may require stabilization to transmit Ryk signaling. We show that the Ryk ICD is degraded following disruption of the Cdc37-Ryk ICD complex during the course of neural differentiation of embryonic stem cells (ESCs). These results suggest a biological role for Cdc37-mediated stabilization of the Ryk ICD in Ryk signaling

EXPERIMENTAL PROCEDURES

RESULTS

Findings

DISCUSSION