PKCβII acts downstream of chemoattractant receptors and mTORC2 to regulate cAMP production and myosin II activity in neutrophils

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 chemoatntractant- 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 C beta II (PKC beta II) by CPG53353 or short hairpin RNA knockdown severely inhibits chemoattractant-induced cAMP synthesis and chemotaxis in neutrophils. Remarkably, PKC beta II-inhibited cells exhibit specific and severe tail retraction defects. In response to chemoattractant stimulation, phosphorylated PKC beta II, but not PKC alpha, is transiently translocated to the plasma membrane, where it phosphorylates and activates AC9. mTORC2-mediated PKC beta II phosphorylation on its turn motif, but not its hydrophobic motif, is required for membrane translocation of PKC beta II. Inhibition of mTORC2 activity by Rictor knockdown not only dramatically decreases PKC beta II activity, but it also strongly inhibits membrane translocation of PKC beta II. Together our findings show that PKC beta II is specifically required for mTORC2-dependent AC9 activation and back retraction during neutrophil chemotaxis.