Mechanisms of plasma membrane targeting of formin mDia2 through its amino terminal domains.
The formin mDia2 mediates the formation of lamellipodia and filopodia during cell locomotion. The subcellular localization of activated mDia2 depends on interactions with actin filaments and the plasma membrane. We investigated the poorly understood mechanism of plasma membrane targeting of mDia2 and found that the entire N-terminal region of mDia2 preceding the actin-polymerizing formin homology domains 1 and 2 (FH1-FH2) module was potently targeted to the membrane. This localization was enhanced by Rif, but not by other tested small GTPases, and depended on a positively charged N-terminal basic domain (BD). The BD bound acidic phospholipids in vitro, suggesting that in vivo it may associate with the plasma membrane through electrostatic interactions. Unexpectedly, a fragment consisting of the GTPase-binding region and the diaphanous inhibitory domain (G-DID), thought to mediate the interaction with GTPases, was not targeted to the plasma membrane even in the presence of constitutively active Rif. Addition of the BD or dimerization/coiled coil domains to G-DID rescued plasma membrane targeting in cells. Direct binding of Rif to mDia2 N terminus required the presence of both G and DID. These results suggest that the entire N terminus of mDia2 serves as a coincidence detection module, directing mDia2 to the plasma membrane through interactions with phospholipids and activated Rif.
Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA.
Roman
R
Gorelik
Changsong
C
Yang
Vasumathi
V
Kameswaran
Roberto
R
Dominguez
Tatyana
T
Svitkina
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D005260:Female; D000080865:Formins; D020558:GTP Phosphohydrolases / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D013329:Structure-Activity Relationship; D020741:rho GTP-Binding Proteins / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; C484571:DIAPH3 protein, human; D000080865:Formins; D020558:GTP Phosphohydrolases; C419867:RHOF protein, human; D020741:rho GTP-Binding Proteins