CMT 4J: ArPIKfyve regulates Sac3 protein abundance and turnover: Disruption of t

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J Biol Chem. 2010 Jul 14

ArPIKfyve regulates Sac3 protein abundance and turnover: Disruption of the

mechanism by Sac3I41T mutation causing Charcot-Marie-Tooth 4J disorder.

Ikonomov OC, Sbrissa D, Fligger J, Delvecchio K, Shisheva A.

Wayne State University School of Medicine, United States.

Abstract

The mammalian PtdIns(3,5)P2 phosphatase Sac3 and ArPIKfyve, the associated

regulator of the PtdIns3P-5 kinase PIKfyve, form a stable binary complex that

associates with PIKfyve in a ternary complex to increase PtdIns(3,5)P2

production. Whether the ArPIKfyve-Sac3 subcomplex functions outside the PIKfyve

context is unknown.

Here we show that stable or transient expression of ArPIKfyveWT in mammalian

cells elevates steady-state protein levels and the PtdIns(3,5)P2-hydrolyzing

activity of Sac3, whereas knockdown of ArPIKfyve has the opposite effect. These

manipulations do not alter the Sac3 mRNA levels, suggesting that ArPIKfyve might

control Sac3 protein degradation.

Inhibition of protein synthesis in COS cells by cycloheximide reveals remarkably

rapid turnover of expressed Sac3WT (t1/2=18.8 min), resulting from a

proteasome-dependent clearance as evidenced by the extended Sac3WT half-life

upon inhibiting proteasome activity. Coexpression of ArPIKfyveWT, but not the N-

or C-terminal halves, prolongs the Sac3WT half-life consistent with enhanced

Sac3 protein stability through association with full-length ArPIKfyve.

We further demonstrate that mutant Sac3, harboring the pathogenic I-to-T

substitution at position 41 found in patients with CMT4J disorder is similar to

Sac3WT with regard to PtdIns(3,5)P2-hydrolyzing activity, association with

ArPIKfyve or rapid proteasome-dependent clearance. Remarkably, however, neither

the steady-state Sac3I41T is elevated nor is the Sac3I41T half-life extended by

coexpressed ArPIKfyveWT indicating that unlike with Sac3WT, ArPIKfyve fails to

prevent Sac3I41T rapid loss.

Together, our data indentify a novel regulatory mechanism whereby ArPIKfyve

enhances Sac3 abundance by attenuating Sac3 proteasome-dependent degradation and

suggest that a failure of this mechanism could be the primary molecular defect

in the pathogenesis of CMT4J.