Mitotic down-regulation of p190RhoGAP is required for the successful completion of cytokinesis.
p190RhoGAP-A (p190) is a GTPase-activating protein known to regulate actin cytoskeleton dynamics by decreasing RhoGTP levels through activation of Rho intrinsic GTPase activity. We have previously shown that p190 protein levels are cell cycle-regulated, decreasing in mitosis, and that this decrease is mediated by the ubiquitin-proteasome pathway. In addition, overexpression of p190 results in decreased RhoGTP levels at the cleavage furrow during cytokinesis, p190 and the RhoGEF Ect2 play opposing roles in cytokinesis, and sustained levels of p190 in mitosis are associated with cytokinesis failure, all findings that suggest but do not directly demonstrate that completion of cytokinesis is dependent on reduced levels of p190. Here we report, using an RNAi reconstitution approach with a degradation-resistant mutant, that decreased p190 levels are required for successful cytokinesis. We also show that the multinucleation phenotype is dependent on p190 RhoGAP activity, determine that the N-terminal GBDS1 region is necessary and sufficient for p190 mitotic ubiquitination and degradation, and identify four N-terminal residues as necessary for the degradation of p190 in mitosis. Our data indicate that in addition to activation of RhoGEF(s), reduction of RhoGAP (p190) is a critical mechanism by which increased RhoGTP levels are achieved in late mitosis, thereby ensuring proper cell division.
Department of Microbiology and Cancer Center, University of Virginia, Charlottesville, Virginia 22908.
Sergio A Sánchez
SAS
Manchinelly
Department of Microbiology and Cancer Center, University of Virginia, Charlottesville, Virginia 22908.
Joyce Agati
JA
Miller
Department of Microbiology and Cancer Center, University of Virginia, Charlottesville, Virginia 22908.
Ling
L
Su
Department of Microbiology and Cancer Center, University of Virginia, Charlottesville, Virginia 22908.
Tsuyoshi
T
Miyake
Department of Pediatrics, University of Virginia, Charlottesville, Virginia 22908.
Lisa
L
Palmer
Department of Microbiology and Cancer Center, University of Virginia, Charlottesville, Virginia 22908.
Masahito
M
Mikawa
Department of Microbiology and Cancer Center, University of Virginia, Charlottesville, Virginia 22908. Electronic address: sap@virginia.edu.
Sarah J
SJ
Parsons
D000199:Actins / Q000235:genetics / Q000378:metabolism; D048749:Cytokinesis / Q000502:physiology*; D003599:Cytoskeleton / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis / Q000502:physiology*; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D064067:Rho Guanine Nucleotide Exchange Factors; D054875:Ubiquitination / Q000502:physiology
C074560:ARHGAP35 protein, human; D000199:Actins; D020662:Guanine Nucleotide Exchange Factors; D012097:Repressor Proteins; D064067:Rho Guanine Nucleotide Exchange Factors