Abstract
Phosphatidylinositol 4-phosphate 5-kinase type I γ (PIPKIγ90) ubiquitination and subsequent degradation regulate focal adhesion assembly, cell migration, and invasion. However, it is unknown how upstream signals control PIPKIγ90 ubiquitination or degradation. Here we show that p70S6K1 (S6K1), a downstream target of mechanistic target of rapamycin (mTOR), phosphorylates PIPKIγ90 at Thr-553 and Ser-555 and that S6K1-mediated PIPKIγ90 phosphorylation is essential for cell migration and invasion. Moreover, PIPKIγ90 phosphorylation is required for the development of focal adhesions and invadopodia, key machineries for cell migration and invasion. Surprisingly, substitution of Thr-553 and Ser-555 with Ala promoted PIPKIγ90 ubiquitination but enhanced the stability of PIPKIγ90, and depletion of S6K1 also enhanced the stability of PIPKIγ90, indicating that PIPKIγ90 ubiquitination alone is insufficient for its degradation. These data suggest that S6K1-mediated PIPKIγ90 phosphorylation regulates cell migration and invasion by controlling PIPKIγ90 degradation.
Highlights
Phosphatidylinositol 4-phosphate 5-kinase type I ␥ (PIPKI␥90) ubiquitination and subsequent degradation regulate focal adhesion assembly, cell migration, and invasion
We show that p70S6K1 (S6K1), a downstream target of mechanistic target of rapamycin, phosphorylates PIPKI␥90 at Thr-553 and Ser-555 and that S6K1-mediated PIPKI␥90 phosphorylation is essential for cell migration and invasion
The ubiquitin proteasome pathway regulates focal adhesions (FAs) assembly and disassembly and, cell migration and invasion by ubiquitinating FA proteins [16, 21,22,23,24,25,26], and we recently demonstrated that PIPKI␥90 ubiquitination and subsequent degradation control FA dynamics to regulate cell migration and invasion [16]
Summary
MDA-MB-231 cells stably expressing FLAG-PIPKI␥90 were serum-starved and stimulated with. FLAG-PIPKI␥90 was immunoprecipitated (IP), and phosphorylation was detected with an anti-RXRXXpS/T motif antibody. MDA-MB-231 cells stably expressing FLAG-PIPKI␥90 were serumstarved, treated with Akt inhibitor VIII and the S6K1 inhibitors DG2 (10 M) or PF4708671 (10 M), and stimulated with HGF for 20 min. S6K1 in MDA-MB-231 cells stably expressing FLAG-PIPKI␥90 was depleted using lentiviruses that express S6K1 shRNAs. Cell were serum-starved and stimulated with HGF (20 ng/ml) for 20 min. We demonstrate that S6K1 phosphorylates PIPKI␥90 at Thr-553 and Ser-555 and that S6K1-mediated phosphorylation controls PIPKI␥90 degradation to regulate the development of FAs and invadopodia and, cell migration and invasion
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