The Dbl-related protein, Lfc, localizes to microtubules and mediates the activation of Rac signaling pathways in cells.
The possibility that the Dbl family member Lfc can activate Rac1 in cells is investigated in this study. Previously, we demonstrated that both Lfc and Lsc, like their closest relative Lbc, can act catalytically in stimulating the guanine nucleotide exchange activity of RhoA in vitro. Neither Lfc nor Lsc stimulated the in vitro exchange activity of Cdc42 or Rac1; however, Lfc was capable of forming a tight complex with Rac1 in vitro. We show here that Lfc stimulates c-Jun kinase (JNK) activity in COS-7 cells. This stimulation was blocked by a dominant negative mutant of Rac1 and somewhat less effectively by dominant negative RhoA, but not by dominant negative Cdc42. Overexpression of Lfc in NIH 3T3 cells induced the formation of actin stress fibers and membrane ruffles, consistent with the activation of both RhoA and Rac1 signaling pathways, whereas overexpression of Lsc led exclusively to well developed stress fibers. Using a recently developed assay for measuring the cellular activation of Rac, we did not find that expression of Lfc increased the levels of GTP-bound Rac1. However, an examination of the cellular localization of Lfc showed that it was localized to microtubules, similar to what has been reported for activated Rac1, the mixed lineage kinase (MLK) and JNK. Moreover, we have found that the Pleckstrin homology (PH) domain of Lfc specifically associates with tubulin. Taken together, these findings suggest a model where the PH domain-mediated localization of Lfc to microtubules enables the recruitment of Rac to a site proximal to its signaling targets, resulting in JNK activation and actin cytoskeletal changes.
Glaven
J A
JA
Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853-6401, USA.
Whitehead
I
I
Bagrodia
S
S
Kay
R
R
Cerione
R A
RA
D016475:3T3 Cells; D000199:Actins / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002846:Chromatography, Affinity; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D048031:JNK Mitogen-Activated Protein Kinases; D051379:Mice; D008870:Microtubules / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases; D009154:Mutation; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D064067:Rho Guanine Nucleotide Exchange Factors; D015398:Signal Transduction; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism; D020744:rac GTP-Binding Proteins
D000199:Actins; C577402:Arhgef2 protein, mouse; D020662:Guanine Nucleotide Exchange Factors; D011518:Proto-Oncogene Proteins; D064067:Rho Guanine Nucleotide Exchange Factors; D014404:Tubulin; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D019204:GTP-Binding Proteins; D020744:rac GTP-Binding Proteins