title: Cdk5 regulates Rap1 activity.
abstract: Rap1 signaling is important for migration, differentiation, axonal growth, and during neuronal polarity. Rap1 can be activated by external stimuli, which in turn regulates specific guanine nucleotide exchange factors such as C3G, among others. Cdk5 functions are also important to neuronal migration and differentiation. Since we found that pharmacological inhibition of Cdk5 by using roscovitine reduced Rap1 protein levels in COS-7 cells and also C3G contains three putative phosphorylation sites for Cdk5, we examined whether the Cdk5-dependent phosphorylation of C3G could affect Rap1 expression and activity. We co-transfected C3G and tet-OFF system for p35 over-expression, an activator of Cdk5 activity into COS-7 cells, and then we evaluated phosphorylation in serine residues in C3G by immunoprecipitation and Western blot. We found that p35 over-expression increased C3G-serine-phosphorylation while inhibition of p35 expression by tetracycline or inhibition of Cdk5 activity with roscovitine decreased it. Interestingly, we found that MG-132, a proteasome inhibitor, rescue Rap1 protein levels in the presence of roscovitine. Besides, C3G-serine-phosphorylation and Rap1 protein levels were reduced in brain from Cdk5(-/-) as compared with the Cdk5(+/+) brain. Finally, we found that p35 over-expression increased Rap1 activity while inhibition of p35 expression by tetracycline or roscovitine decreased Rap1 activity. These results suggest that Cdk5-mediated serine-phosphorylation of C3G may control Rap1 stability and activity, and this may potentially impact various neuronal functions such as migration, differentiation, and polarity.
authors: Laboratory of Cellular and Neuronal Dynamics, Department of Biology, Faculty of Sciences, Universidad de Chile, Chile.
authors: Elias
authors: E
authors: Utreras
authors: Daniel
authors: D
authors: Henriquez
authors: Erick
authors: E
authors: Contreras-Vallejos
authors: Cristina
authors: C
authors: Olmos
authors: Alex
authors: A
authors: Di Genova
authors: Alejandro
authors: A
authors: Maass
authors: Ashok B
authors: AB
authors: Kulkarni
authors: Christian
authors: C
authors: Gonzalez-Billault
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002522:Chlorocebus aethiops; D051360:Cyclin-Dependent Kinase 5 / Q000502:physiology*; D015853:Cysteine Proteinase Inhibitors; D020735:Guanine Nucleotide-Releasing Factor 2 / Q000502:physiology; D047468:Immunoprecipitation; D053765:Interleukin-12 Subunit p35 / Q000096:biosynthesis; D007976:Leupeptins / Q000494:pharmacology; D050576:Membrane Fusion Proteins; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009474:Neurons / Q000187:drug effects; D010766:Phosphorylation; D060888:Real-Time Polymerase Chain Reaction; D013752:Tetracycline / Q000494:pharmacology; D014162:Transfection; D020827:rap1 GTP-Binding Proteins / Q000502:physiology*
chemical_list: D015853:Cysteine Proteinase Inhibitors; D020735:Guanine Nucleotide-Releasing Factor 2; D053765:Interleukin-12 Subunit p35; D007976:Leupeptins; D050576:Membrane Fusion Proteins; D051360:Cyclin-Dependent Kinase 5; D020827:rap1 GTP-Binding Proteins; D013752:Tetracycline; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde