ABSTRACT
Chemotaxis is a process by which cells polarize and move up a chemical gradient through the spatiotemporal regulation of actin assembly and actomyosin contractility, which ultimately control front protrusions and back retractions. We previously demonstrated that in neutrophils, mammalian target of rapamycin complex 2 (mTORC2) is required for chemoattractant-mediated activation of adenylyl cyclase 9 (AC9), which converts ATP into cAMP and regulates back contraction through MyoII phosphorylation. Here we study the mechanism by which mTORC2 regulates neutrophil chemotaxis and AC9 activity. We show that inhibition of protein kinase CßII (PKCßII) by CPG53353 or short hairpin RNA knockdown severely inhibits chemoattractant-induced cAMP synthesis and chemotaxis in neutrophils. Remarkably, PKCßII-inhibited cells exhibit specific and severe tail retraction defects. In response to chemoattractant stimulation, phosphorylated PKCßII, but not PKCα, is transiently translocated to the plasma membrane, where it phosphorylates and activates AC9. mTORC2-mediated PKCßII phosphorylation on its turn motif, but not its hydrophobic motif, is required for membrane translocation of PKCßII. Inhibition of mTORC2 activity by Rictor knockdown not only dramatically decreases PKCßII activity, but it also strongly inhibits membrane translocation of PKCßII. Together our findings show that PKCßII is specifically required for mTORC2-dependent AC9 activation and back retraction during neutrophil chemotaxis.