Autoinhibition of p50 Rho GTPase-activating protein (GAP) is released by prenylated small GTPases.
Interaction of p50 Rho GTPase-activating protein (p50RhoGAP) with Rho family small GTPases was investigated in a yeast two-hybrid system, by radioactive GAP assay, and in a Rac-regulated enzymatic reaction, through superoxide production by the phagocytic NADPH oxidase. The yeast two-hybrid system revealed an interaction between the C-terminal GAP domain and the N-terminal part of p50RhoGAP. The first 48 amino acids play a special role both in the stabilization of the intramolecular interaction and in recognition of the prenyl tail of small GTPases. The GAP assay and the NADPH oxidase activity indicate that the GTPase-activating effect of full-length p50RhoGAP is lower on non-prenylated than on prenylated small GTPase. Removal of amino acids 1-48 and 169-197 of p50RhoGAP increases the GAP effect on non-prenylated Rac, whereas prenylated Rac reacts equally well with the full-length and the truncated proteins. We suggest that p50RhoGAP is in an autoinhibited conformation stabilized by the stretches 1-48 and 169-197 and the prenyl group of the small GTPase plays a role in releasing this intramolecular restraint.
Moskwa
Patryk
P
Department of Physiology, Semmelweis University, H-1444 Budapest, Hungary.
Paclet
Marie-Hélène
MH
Dagher
Marie-Claire
MC
Ligeti
Erzsébet
E
D000595:Amino Acid Sequence; D000818:Animals; D002474:Cell-Free System; D020690:GTPase-Activating Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D019255:NADPH Oxidases / Q000378:metabolism; D011485:Protein Binding; D017368:Protein Prenylation; D017434:Protein Structure, Tertiary; D013481:Superoxides / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
D020690:GTPase-Activating Proteins; C085379:rho GTPase-activating protein; D013481:Superoxides; D019255:NADPH Oxidases; D020559:Monomeric GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein