Abstract

Efficient phagocytosis of apoptotic cells is crucial for many cellular processes. One of earliest signals to the phagocyte is the expression of phosphatidylserine (PS) on the outer surface of the apoptotic cell that provides a potent ‘eat-me’ signal. Mer-family tyrosine kinases and αvβ5(3) integrin are major receptors recognizing PS through the opsonizing proteins MFG-E8, or Gas6, respectively. The previous studies have suggested Mer and integrin αvβ5 signaling pathways converge on tyrosine phosphorylation of FAK and p130Cas, as well as downstream CrkII/Dock180/Rac1 activation, evolutionally-conserved for engulfment. In this study, we aimed at investigating how Mer initiates early signaling events to mediate phaogcytosis and activation of integrin αvβ5, and found that Gas6-Mer-induced tyrosine phosphorylation of FAK and p130Cas was inhibited by calphostin C, and engulfment of apoptotic cells by 293T cells overexpressing wild-type Mer was also suppressed by membrane-permeable PKC inhibitory peptide, suggesting that PKC activation is an early signaling event in Mer-mediated phagocytosis. Consistently, we found that Mer activation stimulates tyrosine phosphorylation of PLCγ2, an upstream event of PKC activation. More interestingly, using constitutively active form for Mer (CDMer) and CDMer mutants, we further found that Tyr 867 of Mer is predominantly responsible for PLCγ2 phosphorylation. In addition, Mer activation stimulated complex formation between integrin αvβ5 and Rack1, a receptor for activated PKC. Taken together, PKC is required for Mer-mediated activation signals for phagocytosis, and might be involved in the crosstalk between Mer and integirn αvβ5 via interaction with Rack1.

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