Regulation of the Rac1-specific exchange factor Tiam1 involves both phosphoinositide 3-kinase-dependent and -independent components.
The small GTPase Rac1 is involved in regulating membrane ruffling, gene transcription, cell-cycle progression and cell transformation, and some of these events are blocked by inhibitors of phosphoinositide 3-kinase (PI 3-kinase). Moreover, Rac1 can be activated by several guanine nucleotide exchange factors, which facilitate the release of GDP. We therefore investigated the ability of PI 3-kinase lipid products to regulate Tiam1, a Rac1-specific exchange factor. Tiam1 bound to polyphosphorylated inositol lipids in the rank order PtdIns(3,4,5)P(3)>PtdIns(3,4)P(2) >>PtdIns(4,5)P(2), and this binding could be attributed to the N-terminal pleckstrin-homology (N-PH) domain. Both PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) enhanced Tiam1 guanine nucleotide exchange activity in vitro, but PtdIns(4,5)P(2) had no effect. Co-expression of a constitutively active PI 3-kinase with Tiam1 increased the amount of GTP-bound Rac1 in vivo, a response which required the N-PH domain of Tiam1. Ectopic expression of Tiam1 caused membrane ruffling in Swiss 3T3 cells that was characterized by wortmannin-sensitive and -insensitive components, which required the N-PH domain and the C-terminal PH domain of Tiam1 respectively. These results reveal novel facets of Tiam1-dependent regulation of Rac1 function.
Fleming
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Department of Biochemistry, Medical Sciences Institute, University of Dundee, Dundee DD1 5EH, Scotland, UK. I.N.Fleming@Dundee.ac.uk
Gray
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A
Downes
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CP
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D051379:Mice; D009154:Mutation / Q000235:genetics; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D000075684:T-Lymphoma Invasion and Metastasis-inducing Protein 1; D000077191:Wortmannin; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
D000730:Androstadienes; D001798:Blood Proteins; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D019869:Phosphatidylinositol 3-Kinases; D010716:Phosphatidylinositols; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins; D011506:Proteins; D000075684:T-Lymphoma Invasion and Metastasis-inducing Protein 1; D000077191:Wortmannin; D020830:rac1 GTP-Binding Protein; C087334:TIAM1 protein, human; C513260:Tiam1 protein, mouse; C039077:platelet protein P47