Cellular distribution and functions of wild-type and constitutively activated Dictyostelium PakB.
Dictyostelium PakB, previously termed myosin I heavy chain kinase, is a member of the p21-activated kinase (PAK) family. Two-hybrid assays showed that PakB interacts with Dictyostelium Rac1a/b/c, RacA (a RhoBTB protein), RacB, RacC, and RacF1. Wild-type PakB displayed a cytosolic distribution with a modest enrichment at the leading edge of migrating cells and at macropinocytic and phagocytic cups, sites consistent with a role in activating myosin I. PakB fused at the N terminus to green fluorescent protein was proteolyzed in cells, resulting in removal of the catalytic domain. C-terminal truncated PakB and activated PakB lacking the p21-binding domain strongly localized to the cell cortex, to macropinocytic cups, to the posterior of migrating cells, and to the cleavage furrow of dividing cells. These data indicate that in its open, active state, the N terminus of PakB forms a tight association with cortical actin filaments. PakB-null cells displayed no significant behavioral defects, but cells expressing activated PakB were unable to complete cytokinesis when grown in suspension and exhibited increased rates of phagocytosis and pinocytosis.
de la Roche
Marc
M
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Mahasneh
Amjad
A
Lee
Sheu-Fen
SF
Rivero
Francisco
F
Côté
Graham P
GP
D000199:Actins / Q000737:chemistry; D000818:Animals; D015139:Blotting, Southern; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000096:biosynthesis* / Q000502:physiology*; D020134:Catalytic Domain; D002465:Cell Movement; D048749:Cytokinesis; D004247:DNA / Q000378:metabolism; D004023:Dictyostelium; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D008853:Microscopy; D008957:Models, Genetic; D024461:Myosin Type I / Q000378:metabolism; D010587:Phagocytosis; D010641:Phenotype; D010873:Pinocytosis; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis* / Q000378:metabolism / Q000502:physiology*; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D020798:Two-Hybrid System Techniques; D054462:p21-Activated Kinases; D020741:rho GTP-Binding Proteins / Q000378:metabolism
D000199:Actins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004247:DNA; C515903:PAK1 protein, human; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C033142:myosin-heavy-chain kinase; D024461:Myosin Type I; D020741:rho GTP-Binding Proteins