IQGAP1 promotes neurite outgrowth in a phosphorylation-dependent manner.
In eukaryotic cells IQGAP1 binds to and alters the function of several proteins, including actin, E-cadherin, beta-catenin, Cdc42, and Rac1. Yeast IQGAP1 homologues have an important role in cytoskeletal organization, suggesting that modulation of the cytoskeleton is a fundamental role of IQGAP1. Phosphorylation is a common mechanism by which cells regulate protein function. Here we demonstrate that endogenous IQGAP1 is highly phosphorylated in MCF-7 human breast epithelial cells. Moreover, incubation of cells with phorbol 12-myristate 13-acetate (PMA) stimulated phosphate incorporation into IQGAP1. By using mass spectrometry, Ser-1443 was identified as the major site phosphorylated on IQGAP1 in intact cells treated with PMA. Ser-1441 was also phosphorylated but to a lesser extent. In vitro analysis with purified proteins documented that IQGAP1 is a substrate for protein kinase Cepsilon, which catalyzes phosphorylation on Ser-1443. Consistent with these findings, inhibition of cellular protein kinase C via bisindolymaleimide abrogated Ser-1443 phosphorylation in response to PMA. To elucidate the biological sequelae of phosphorylation, Ser-1441 and Ser-1443 were converted either to alanine, to create a nonphosphorylatable construct, or to glutamic acid and aspartic acid, respectively, to generate a phosphomimetic IQGAP1. Although overexpression of wild type IQGAP1 promoted neurite outgrowth in N1E-115 neuroblastoma cells, the nonphosphorylatable IQGAP1 S1441A/S1443A had no effect. In contrast, the S1441E/S1443D mutation markedly enhanced the ability of IQGAP1 to induce neurite outgrowth. Our data disclose that IQGAP1 is phosphorylated at multiple sites in intact cells and that phosphorylation of IQGAP1 will alter its ability to regulate the cytoskeleton of neuronal cells.
Li
Zhigang
Z
Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
McNulty
Dean E
DE
Marler
Katharine J M
KJ
Lim
Louis
L
Hall
Christine
C
Annan
Roland S
RS
Sacks
David B
DB
D000595:Amino Acid Sequence; D000818:Animals; D001943:Breast Neoplasms; D045744:Cell Line, Tumor; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005260:Female; D006801:Humans; D007211:Indoles; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D008301:Maleimides; D008969:Molecular Sequence Data; D016501:Neurites / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051744:Protein Kinase C-epsilon; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism; D020703:ras GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*
C101203:IQ motif containing GTPase activating protein 1; D007211:Indoles; D007527:Isoenzymes; D008301:Maleimides; D011993:Recombinant Fusion Proteins; D020703:ras GTPase-Activating Proteins; D012694:Serine; C497257:Prkce protein, rat; C497255:PRKCE protein, human; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon; C088060:bisindolylmaleimide; D013755:Tetradecanoylphorbol Acetate