IQGAP1 Promotes Neurite Outgrowth in a Phosphorylation-dependent Manner

Abstract

In eukaryotic cells IQGAP1 binds to and alters the function of several proteins, including actin, E-cadherin, ␤-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 C⑀, 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.Initially identified 10 years ago, IQGAP1 has been shown to participate in several fundamental cellular processes (for reviews see Refs 1 and 2). These include cell-cell attachment, ␤-catenin-mediated transcription, cell migration, regulation of actin, microtubule function, the mitogen-activated protein kinase cascade, and Ca 2ϩ /calmodulin signaling (2-4). IQGAP1 is a component of these diverse functions via direct interactions with multiple target proteins that are mediated by a number of protein interaction motifs in IQGAP1. These include the following: a calponin homology domain, responsible for actin binding (5, 6); a WW motif, which is necessary for the association of extracellular signal-regulated kinase 2 (a component of the mitogen-activated protein kinase pathway) (4); a calmodulinbinding IQ domain (5, 7); and a RasGAP-related domain that binds the small GTPases Cdc42 and Rac1 (5, 8). In addition, IQGAP1 binds to and regulates the functions of E-cadherin, ␤-catenin, and CLIP-170 (9 -12).Accumulating evidence reveals an important role for IQGAP1 in cytoskeletal function. The cytoskeleton of eukaryotic cells comprises several elements, including actin, microtubules, and intermediate filaments. Numerous proteins interact with the actin cytoskeleton...

Multiple proteins mediate IQGAP1-stimulated cell migration

“…This is not surprising as IQGAP1 is a significant modulator of cytoskeletal architecture across a wide spectrum of organisms, ranging from yeast to Xenopus to mammals [15]. IQGAP1 influences numerous cell functions, including actin polymerization, microtubule function, cell-cell adhesion and neurite outgrowth [3,15,16,35]. These effects are mediated by a direct interaction between IQGAP1 and multiple-binding proteins [3,16].…”

MAPK Scaffold IQGAP1 Binds the EGF Receptor and Modulates Its Activation

“…We and others have previously shown that IQGAP1 is phosphorylated in cells in a PKC⑀-dependent manner at Ser 1443 in response to phorbol ester treatment (28,37). It seems reasonable, therefore, to hypothesize that IQGAP1 can be phosphorylated on Ser 1443 in response to EGF-stimulated activation of EGFR.…”

“…IQGAP1-IQ (amino acids 717-916) and IQGAP1⌬IQ (full length minus amino acids 746 -860) have been described previously (27). IQGAP1-SAA (serines 1441 and 1443 mutated to alanine) and IQGAP1-SED (serines 1441 and 1443 mutated to glutamic acid and aspartic acid, respectively), were generated as Myc-tagged constructs and have been described previously (28). Glutathione S-transferase-tagged full-length IQGAP1 fusion protein (GST-IQGAP1) has been described previously (26).…”

“…EGFR triggers phospholipase C␥ to generate both diacylglycerol and inositol 1,4,5-trisphosphate-mediated calcium fluxes, resulting in the potential activation of multiple PKC isoforms (40,41). Previous studies have shown that Ser 1443 is contained within a PKC⑀ consensus site and that PKC⑀ is able to catalyze IQGAP1 phosphorylation at this site in vitro (28,37). To further characterize PKC involvement in EGF-mediated phosphorylation of IQGAP1, we performed targeted knockdown of the TPA-responsive PKC isoenzymes expressed in HeLa cells (42).…”

“…Enzymatic digestion of the purified IQGAP1 with trypsin releases peptides containing the Ser 1443 epitope (28). To measure the abundance of both phosphorylated and nonphosphorylated epitopes, we developed a targeted, full-time LC-MS/MS experiment that rapidly cycles between the four Ser 1443 epitopes produced by trypsin digestion of IQGAP1 (see Fig.…”