ArPIKfyve Regulates Sac3 Protein Abundance and Turnover

Abstract

The mammalian phosphatidylinositol (3,5)-bisphosphate (PtdIns(3,5)P 2 ) 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)P 2 production. I41T 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.The phosphatase Sac3 and the kinase PIKfyve 2 are responsible for synthesis and turnover of phosphatidylinositol (PtdIns) (3,5)P 2 in mammalian cells (1). Intriguingly, both the endogenous and the ectopically expressed enzymes are found to reside in the same regulatory complex, called the PAS complex (for PIKfyve-ArPIKfyve-Sac3), organized by the PIKfyve regulator ArPIKfyve and its ability to homooligomerize (2, 3). Despite their antagonistic activities, PIKfyve and Sac3 appear to be enzymatically active in the ternary complex. Thus, in the case of PIKfyve, formation of the PAS regulatory core is critical for PIKfyve activation (3). Likewise, the Sac3 phosphatase retains its PtdIns(3,5)P 2 -hydrolyzing activity within the PAS ternary complex (4). These data reveal an unusual paradigm whereby a common complex relays two opposing activities, one for synthesis, another for degradation, the physiological meaning of which is yet to be understood (1, 5). Data from in vitro reconstitution studies indicate increased and decreased PtdIns(3,5)P 2 levels triggering mammalian endosome fission and fusion, respectively (2, 6). Thus, an association of the two active yet antagonistic enzymes in a common complex would be consistent with the critical requirement for a tight control of PtdIns(3,5)P 2 homeostasis related to dynamic endosome membrane remodeling through fission and fusion (1). Understanding the spatial and temporal regulation and the coordination of the individual enzyme activities within the PAS complex is essential in providing a better comprehension of the intricate PtdIns(3,5)P 2 homeostatic mechanism. Maintaining PtdIns(3,5)P 2 homeostasis is apparently indispensable for life as evidenced by the early lethality of Drosophila melanogaster or Caenorhabditis elegans PIKfyve-null mutants (7,8) and by the early death of ArPIKfyve and Sac3-deficient mouse models (9, 10). Concordantly, a defective Sac3 I41T allele in combination with a null allele is responsible for the pathogenesis of Charcot-Marie-Tooth type 4J (CMT4J) peripheral neuropathy, a recessively inherited disease with early onset manifested by progressive motor and sensory neuron degeneration (10, 11). The molecular and cellular mechanisms rendering this single I41T amino acid substitution pathogenic are currently unknown.ArPIKfyve associates with the Sac3 phosphatase independently of PIKfyve in a stable ArPIKfyve-Sac3 heterooligomer (3,4). This binary association is apparently a prerequisite for a produc...

Charcot Marie Tooth disease type 4J with complex central nervous system features

“…The unaffected father was found to have both the FIG4 intronic and the LITAF variants – excluding the LITAF mutation as pathogenic. The FIG4 c.122T>C variant (I41T) is one of the most common variants in patients with CMT4J, and leads to rapid degradation of the FIG4 proteins 4. The intronic FIG4 variant was recently reported in a person with CMT4J, also with the I41T variant5 and was absent in >60,000 exomes [exac.broadinstitute.org].…”

Phosphoinositides, Major Actors in Membrane Trafficking and Lipid Signaling Pathways

“…Furthermore, the PtdIns(3,5) P 2 synthesis pathway described in yeast is conserved in human, with PIKfyve interacting with multiple partners [108]. This interaction is indirect and requires the adapter protein ArPIKfyve/VAC14 to stabilize the complex and stimulate PIKfyve activity [109]. As in yeast cells, VAC14 serves as a platform regulating PtdIns(3,5) P 2 synthesis by interacting directly with PIKfyve, FIG4/SAC3 and VAC7 to fine tune the regulation of PtdIns(3,5) P 2 levels [109,110].…”

“…This interaction is indirect and requires the adapter protein ArPIKfyve/VAC14 to stabilize the complex and stimulate PIKfyve activity [109]. As in yeast cells, VAC14 serves as a platform regulating PtdIns(3,5) P 2 synthesis by interacting directly with PIKfyve, FIG4/SAC3 and VAC7 to fine tune the regulation of PtdIns(3,5) P 2 levels [109,110]. Mutation in the FIG4 gene causes Charcot-Marie-Tooth neuropathy [111], and the Mtmr2 knock-out mice model for the disease has elevated PtdIns(3,5) P 2 levels [112].…”

Mouse Models of PI(3,5)P2 Deficiency with Impaired Lysosome Function

“…Further analysis demonstrated that the instability of the I41T protein is a consequence of impaired binding to the VAC14 scaffold protein. The level of FIG4-I41T is also very low in patient fibroblasts, and can be increased by inhibition of degradation by the proteasome pathway (Lenk et al, 2011; Ikonomov et al, 2010). …”