Induction of lamellipodia by Kalirin does not require its guanine nucleotide exchange factor activity
Guanine nucleotide exchange factor (GEF) domains of the Dbl family occur in a variety of proteins that include multiple protein-protein and protein-lipid interaction domains. We used an epithelial-derived cell line to investigate the mechanisms by which the two GEF domains of Kalirin, a neuronal Rho GEF, influence morphology. As expected, Kal-GEF1, an efficient GEF for Rac1 and RhoG, induced the formation of lamellipodia resembling those induced by active Rac1. Although Kal-GEF1 activated Rac and Pak, its ability to induce formation of lamellipodia was not blocked by dominant negative Rho GTPases or by catalytically inactive Pak. Consistent with this, a catalytically inactive mutant of Kal-GEF1 induced formation of lamellipodia and activated Pak. Active Pak was required for the GEF-activity independent effect of Kal-GEF1 and the lamellipodia produced were filled with ribs of filamentous actin. Kal-GEF1 and a GEF-dead mutant co-immunoprecipitated with Pak. The interaction of Kal-GEF1 with Pak is indirect and requires the regulatory protein binding domain of Pak. Filamin A, which is known to interact with and activate Pak, binds to both catalytically active and inactive Kal-GEF1, providing a link by which catalytically inactive Kal-GEF1 can activate Pak and induce lamellipodia. Together, our results indicate that Kal-GEF1 induces lamellipodia through activation of Pak, where GEF activity is not required. GEF-activity-independent effects on downstream targets may be a general property of RhoGEFs.
Actin genes; Actins/metabolism; Cell lines; Cell Shape/physiology; Cells—Morphology; Contractile Proteins/metabolism; Dbl Family; Filamins; G proteins; Genetics; GTP Phosphohydrolases/genetics; Guanine Nucleotide Exchange Factors/genetics; Guanine Nucleotide Exchange Factors/metabolism; Guanine Nucleotide Exchange Factors/physiology; Guanosine triphosphatase; Histones; Histones/metabolism; Humans; Microfilament proteins; Microfilament Proteins/metabolism; Models; Biological; Mutation (Biology); Mutation/genetics; Nerve tissue proteins; Nerve Tissue Proteins/metabolism; Peptide Fragments/genetics; Peptide Fragments/metabolism; Phosphorylation; Protein; Protein binding; Protein Isoforms/genetics; Protein Isoforms/metabolism; Protein-Serine-Threonine Kinases/genetics; Protein-Serine-Threonine Kinases/metabolism; Protein-Serine-Threonine Kinases/physiology; Pseudopodia/physiology; Transfection; Trio; UNC-73; Wiskott-Aldrich Syndrome Protein Family; Wiskott-Aldrich Syndrome Protein; Neuronal; p21-Activated Kinases; rac1 GTP-Binding Protein/genetics; rac1 GTP-Binding Protein/metabolism; rho GTP-Binding Proteins
Cell and Developmental Biology | Life Sciences | Neurosciences
Schiller, M. R.,
Mains, R. E.,
Eipper, B. A.
Induction of lamellipodia by Kalirin does not require its guanine nucleotide exchange factor activity.
Experimental Cell Research, 307(2),