Edited by Alex TokerPhosphatidylinositol 4-phosphate 5-kinase type I ␥ (PIPKI␥90) ubiquitination and subsequent degradation regulate focal adhesion assembly, cell migration, and invasion. However, it is unknown how upstream signals control PIPKI␥90 ubiquitination or degradation. Here we show that p70S6K1 (S6K1), a downstream target of mechanistic target of rapamycin (mTOR), phosphorylates PIPKI␥90 at Thr-553 and Ser-555 and that S6K1-mediated PIPKI␥90 phosphorylation is essential for cell migration and invasion. Moreover, PIPKI␥90 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 PIPKI␥90 ubiquitination but enhanced the stability of PIPKI␥90, and depletion of S6K1 also enhanced the stability of PIPKI␥90, indicating that PIPKI␥90 ubiquitination alone is insufficient for its degradation. These data suggest that S6K1-mediated PIPKI␥90 phosphorylation regulates cell migration and invasion by controlling PIPKI␥90 degradation.Cell migration and invasion are prerequisites for cancer metastasis (1, 2). Thus, the elucidation of the molecular mechanisms of cell migration and invasion is a compelling goal in cancer cell biology.Phosphatidylinositol 4 phosphate 5-kinase type I ␥ (PIPKI␥90) 2 binds talin and localizes to focal adhesions (FAs) (3, 4). It catalyzes ATP-dependent phosphorylation of phosphatidylinositol 4-phosphate (PIP) to generate phosphatidylinositol 4,5-bisphosphate (PIP 2 ), which binds and activates talin, vinculin, and focal adhesion kinase to mediate FA assembly (5, 6). PIP 2 also binds many cytoskeletal proteins, such as neural Wiskott-Aldrich Syndrome protein, gelsolin, and profilin, to regulate actin polymerization (7-10).In addition, PIP 2 is a precursor of several lipid second messengers, such as phosphatidylinositol 3,4,5-triphosphate (PIP 3 ), inositol 1,4,5-triphosphate, and diacylglycerol. We have shown that depletion of PIPKI␥90 completely abolishes PIP 3 production in HCT119 human colon cancer cells (11), indicating a critical role of PIPKI␥90 in lipid signaling. PIPKI␥90 is necessary for epithelial cell adherens junction assembly and progression through the E-cadherin--catenin signal pathway (12). PIPKI␥90 depletion inhibits cell proliferation, MMP9 secretion, and cell motility (13,14). PIPKI␥90 is essential for cell migration, invasion, and metastasis. It is required for focal adhesion assembly and disassembly, key steps in cell migration (11). Depletion of PIPKI␥90 inhibits growth factor-stimulated cell migration in MDA-MB-231 breast cancer cells and HeLa cervical cancer cells (14, 15). PIPKI␥90 knockdown also blocks the invasion of breast cancer and colon cancer cells (11, 16). Furthermore, PIPKI␥90-depleted 4T1 breast cancer cells show significant reduction in tumor progression and metastasis (13). PIPKI␥90 also regulates neutrophil migration by controlling cell polarity as well as rear retraction (17)(18)(19). PIPKI␥90 is a substrate for ...