Cyclin-dependent kinase 5 is a calmodulin-binding protein that associates with puromycin-sensitive aminopeptidase in the nucleus of Dictyostelium.
Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase that has been implicated in a number of cellular processes. In Dictyostelium, Cdk5 localizes to the nucleus and cytoplasm, interacts with puromycin-sensitive aminopeptidase A (PsaA), and regulates endocytosis, secretion, growth, and multicellular development. Here we show that Cdk5 is a calmodulin (CaM)-binding protein (CaMBP) in Dictyostelium. Cdk5, PsaA, and CaM were all present in isolated nuclei and Cdk5 and PsaA co-immunoprecipitated with nuclear CaM. Although nuclear CaMBPs have previously been identified in Dictyostelium, the detection of CaM in purified nuclear fractions had not previously been shown. Putative CaM-binding domains (CaMBDs) were identified in Cdk5 and PsaA. Deletion of one of the two putative CaMBDs in Cdk5 ((132)LLINRKGELKLADFGLARAFGIP(154)) prevented CaM-binding indicating that this region encompasses a functional CaMBD. This deletion also increased the nuclear distribution of Cdk5 suggesting that CaM regulates the nucleocytoplasmic transport of Cdk5. A direct binding between CaM and PsaA could not be determined since deletion of the one putative CaMBD in PsaA prevented the nuclear localization of the deletion protein. Together, this study provides the first direct evidence for nuclear CaM in Dictyostelium and the first evidence in any system for Cdk5 being a CaMBP.
University of Toronto, Department of Cell & Systems Biology, Ontario, Canada. rhuber@chgr.mgh.harvard.edu
Robert J
RJ
Huber
Andrew
A
Catalano
Danton H
DH
O'Day
D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000953:Antigens, Protozoan / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D002467:Cell Nucleus / Q000201:enzymology / Q000378:metabolism*; D051360:Cyclin-Dependent Kinase 5 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D004023:Dictyostelium / Q000201:enzymology / Q000254:growth & development / Q000378:metabolism / Q000648:ultrastructure; D004352:Drug Resistance, Microbial; D047468:Immunoprecipitation; D008562:Membrane Glycoproteins / Q000378:metabolism; D008954:Models, Biological; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000235:genetics / Q000502:physiology; D015800:Protozoan Proteins / Q000378:metabolism; D011691:Puromycin / Q000494:pharmacology; D017384:Sequence Deletion; D014344:Trypanocidal Agents / Q000494:pharmacology
D000953:Antigens, Protozoan; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; D014344:Trypanocidal Agents; C074496:prespore-specific antigen, Dictyostelium discoideum; D011691:Puromycin; D051360:Cyclin-Dependent Kinase 5; D000626:Aminopeptidases