Interaction of PDZRhoGEF with microtubule-associated protein 1 light chains: link between microtubules, actin cytoskeleton, and neuronal polarity.
Rat (r) PDZRhoGEF, initially identified as a glutamate transporter EAAT4-associated protein, is a member of a novel RhoGEF subfamily. The N terminus of the protein contains a PDZ and a proline-rich domain, two motifs known to be involved in protein-protein interactions. By using the yeast two-hybrid approach, we screened for proteins that interact with the N terminus of rPDZRhoGEF. The light chain 2 of microtubule-associated protein 1 (LC2) was the only protein identified from the screen that does not contain a type I PDZ-binding motif at its extreme C terminus (-(S/T)Xphi-COOH, where phi is a hydrophobic amino acid). However, the C terminus does conform to a type II-binding motif (-phiXphi). We report here that rPDZRhoGEF interacts with LC2 via the PDZ domain, and the interaction is abolished by mutations in the carboxylate-binding loop. The specificity of the interaction was confirmed using GST fusion protein pull-down assays and coimmunoprecipitations. Expression of rPDZRhoGEF mutants that are unable to interact with proteins via the carboxylate-binding loop induced changes in cell morphology and actin organization. These mutants alter the activation of RhoGTPases, and coexpression of dominant-negative RhoGTPases prevent the morphological changes. Furthermore, in cells expressing wild type rPDZRhoGEF, drug-induced microtubule depolymerization produces changes in cell morphology that are similar to those induced by rPDZRhoGEF mutants. These results indicate that modulation of the guanine nucleotide exchange activity of rPDZRhoGEF through interaction with microtubule-associated protein light chains may coordinate microtubule integrity and the reorganization of actin cytoskeleton. This coordinated action of the actin and microtubular cytoskeletons is essential for the development and maintenance of neuronal polarity.
Centre for Neuroscience Research, the University of Edinburgh, Edinburgh EH9 1QH, Scotland, United Kingdom.
David M
DM
Longhurst
Mitsunori
M
Watanabe
Jeffrey D
JD
Rothstein
Mandy
M
Jackson
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D016764:Cell Polarity; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques; D020742:rhoA GTP-Binding Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D000199:Actins; D008869:Microtubule-Associated Proteins; D011994:Recombinant Proteins; D020742:rhoA GTP-Binding Protein