A PKC-MARCKS-PI3K regulatory module links Ca(2+) and PIP(3) signals at the leading edge of polarized macrophages

The leukocyte chemosensory pathway detects attractant gradients and directs cell migration to sites of inflammation, infection, tissue damage, and carcinogenesis. Previous studies have revealed that local Ca(2+) and PIP(3) signals at the leading edge of polarized leukocytes play central roles in positive feedback loop essential to cell polarization and chemotaxis. These prior studies showed that stimulation of the leading edge Ca(2+) signal can strongly activate PI3K, thereby triggering a larger PIP(3) signal, but did not elucidate the mechanistic link between Ca(2+) and PIP(3) signaling. A hypothesis explaining this link emerged, postulating that Ca(2+)-activated PKC displaces the MARCKS protein from plasma membrane PIP(2), thereby releasing sequestered PIP(2) to serve as the target and substrate lipid of PI3K in PIP(3) production. In vitro single molecule studies of the reconstituted pathway on lipid bilayers demonstrated the feasibility of this PKC-MARCKS-PI3K regulatory module linking Ca(2+) and PIP(3) signals in the reconstituted system. The present study tests the model predictions in live macrophages by quantifying the effects of: (a) two pathway activators—PDGF and ATP that stimulate chemoreceptors and Ca(2+) influx, respectively; and (b) three pathway inhibitors—wortmannin, EGTA, and Go6976 that inhibit PI3K, Ca(2+) influx, and PKC, respectively; on (c) four leading edge activity sensors—AKT-PH-mRFP, CKAR, MARCKSp-mRFP, and leading edge area that report on PIP(3) density, PKC activity, MARCKS membrane binding, and leading edge expansion/contraction, respectively. The results provide additional evidence that PKC and PI3K are both essential elements of the leading edge positive feedback loop, and strongly support the existence of a PKC-MARCKS-PI3K regulatory module linking the leading edge Ca(2+) and PIP(3) signals. As predicted, activators stimulate leading edge PKC activity, displacement of MARCKS from the leading edge membrane and increased leading edge PIP(3) levels, while inhibitors trigger the opposite effects. Comparison of the findings for the ameboid chemotaxis of leukocytes with recently published findings for the mesenchymal chemotaxis of fibroblasts suggests that some features of the emerging leukocyte leading edge core pathway (PLC-DAG-Ca(2+)-PKC-MARCKS-PIP(2)-PI3K-PIP(3)) may well be shared by all chemotaxing eukaryotic cells, while other elements of the leukocyte pathway may be specialized features of these highly optimized, professional gradient-seeking cells. More broadly, the findings suggest a molecular mechanism for the strong links between phospho-MARCKS and many human cancers.