Phosphorylation of NG2 proteoglycan by protein kinase C-alpha regulates polarized membrane distribution and cell motility.
Protein kinase C (PKC)-alpha 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 alpha(3)beta(1) 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-alpha-mediated NG2 phosphorylation at Thr(2256) is therefore a key step for initiating cell polarization and motility.
Makagiansar
Irwan T
IT
Cancer Research Center, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA. irwanm@burnham.org
Williams
Scott
S
Dahlin-Huppe
Kimberlee
K
Fukushi
Jun-ichi
J
Mustelin
Tomas
T
Stallcup
William B
WB
D000818:Animals; D000941:Antigens / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D002465:Cell Movement; D003593:Cytoplasm / Q000378:metabolism; D003598:Cytoskeletal Proteins; D018698:Glutamic Acid / Q000737:chemistry; D006801:Humans; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D009154:Mutation; D010703:Phorbol Esters / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D017434:Protein Structure, Tertiary; D011509:Proteoglycans / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D013912:Threonine / Q000737:chemistry; D014633:Valine / Q000737:chemistry; D000099216:Chondroitin Sulfate Proteoglycan 4; D000099149:Ezrin
D000941:Antigens; D003598:Cytoskeletal Proteins; D018698:Glutamic Acid; D010703:Phorbol Esters; D010750:Phosphoproteins; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D011509:Proteoglycans; D013912:Threonine; D014633:Valine; D000099216:Chondroitin Sulfate Proteoglycan 4; D000099149:Ezrin; C496628:PRKCA protein, human