Phosphorylation of NG2 proteoglycan by protein kinase C-α regulates polarized membrane distribution and cell motility

Protein Kinase C (PKC)-α phosphorylation of recombinant NG2 cytoplasmic domain and phorbol ester-induced PKC-dependent phosphorylation of full-length NG2 expressed in U251 cells are both blocked by mutation of Thr ²²⁵⁶, identifying this residue as a primary phosphorylation site. In untreated U251/NG2 cells, NG2 is present along with ezrin and α₃β₁ integrin in apical cell surface protrusions. Phorbol ester treatment causes redistribution of all three components to lamellipodia, accompanied by increased cell motility. U251 cells expressing NG2 with a valine substitution at position 2256 are resistant to phorbol ester treatment: NG2 remains in membrane protrusions and cell motility is unchanged. In contrast, NG2 with a glutamic acid substitution at position 2256 redistributes to lamellipodia even without phorbol ester treatment, rendering transfected U251 cells spontaneously motile. PKC-α-mediated NG2 phosphorylation at Thr²²⁵⁶ is therefore a key step for initiating cell polarization and motility.