A plant-specific subclass of C-terminal kinesins contains a conserved a-type cyclin-dependent kinase site implicated in folding and dimerization.
Cyclin-dependent kinases (CDKs) control cell cycle progression through timely coordinated phosphorylation events. Two kinesin-like proteins that interact with CDKA;1 were identified and designated KCA1 and KCA2. They are 81% identical and have a similar three-partite domain organization. The N-terminal domain contains an ATP and microtubule-binding site typical for kinesin motors. A green fluorescent protein (GFP) fusion of the N-terminal domain of KCA1 decorated microtubules in Bright Yellow-2 cells, demonstrating microtubule-binding activity. During cytokinesis the full-length GFP-fusion protein accumulated at the midline of young and mature expanding phragmoplasts. Two-hybrid analysis and coimmunoprecipitation experiments showed that coiled-coil structures of the central stalk were responsible for homo- and heterodimerization of KCA1 and KCA2. By western-blot analysis, high molecular mass KCA molecules were detected in extracts from Bright Yellow-2 cells overproducing the full-length GFP fusion. Treatment of these cultures with the phosphatase inhibitor vanadate caused an accumulation of these KCA molecules. In addition to dimerization, interactions within the C-terminally located tail domain were revealed, indicating that the tail could fold onto itself. The tail domains of KCA1 and KCA2 contained two adjacent putative CDKA;1 phosphorylation sites, one of which is conserved in KCA homologs from other plant species. Site-directed mutagenesis of the conserved phosphorylation sites in KCA1 resulted in a reduced binding with CDKA;1 and abolished intramolecular tail interactions. The data show that phosphorylation of the CDKA;1 site provokes a conformational change in the structure of KCA with implications in folding and dimerization.
Vanstraelen
Marleen
M
Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology, Ghent University, B-9052 Gent, Belgium.
Torres Acosta
Juan Antonio
JA
De Veylder
Lieven
L
Inzé
Dirk
D
Geelen
Danny
D
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018844:Cyclin-Dependent Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D019281:Dimerization; D005822:Genetic Vectors; D016547:Kinesins / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D059828:Plant Cells; D010944:Plants / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis / Q000235:genetics; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000235:genetics
D017931:DNA Primers; D010446:Peptide Fragments; D011994:Recombinant Proteins; D018844:Cyclin-Dependent Kinases; D016547:Kinesins