The Cdk5 Homologue, Crp, Regulates Endocytosis and Secretion in Dictyostelium and Is Necessary for Optimum Growth and Differentiation

Department of Biochemistry and Cell Biology , Rice University, Houston, Texas, United States
Developmental Biology (Impact Factor: 3.55). 08/2002; 247(1):1-10. DOI: 10.1006/dbio.2002.0684
Source: PubMed


Dictyostelium Crp is a member of the cyclin-dependent kinase (Cdk) family of proteins. It is most related in sequence to mammalian Cdk5, which unlike other members of the family, has functions that are unrelated to the cell cycle. In order to better understand the function of Crp in Dictyostelium, we overexpressed a dominant negative form, Crp-D144N, under the control of the actin 15 promoter. Cells overexpressing Crp-D144N exhibit a reduced growth rate in suspension culture and reduced rates of fluid-phase endocytosis and phagocytosis. There is no reduction in Cdc2 kinase activity in extracts from cells overexpressing Crp-D144N, suggesting that the growth defect is not due to inhibition of Cdc2. In addition to the growth defect, the act15::crp-D144N transformants aggregate at a slower rate than wild-type cells and form large aggregation streams. These eventually break up to form small aggregates and most of these do not produce mature fruiting bodies. The aggregation defect is fully reversed in the presence of wild-type cells but terminal differentiation is only partially rescued. In act15::crp-D144N transformants, the countin component of the counting factor, a secreted protein complex that regulates the breakup of streams, mostly appears outside the cell as degradation products and the reduced level of the intact protein may at least partially account for the initial formation of the large aggregation streams. Our observations indicate that Crp is important for both endocytosis and efflux and that defects in these functions lead to reduced growth and aberrant development.

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    • "Interestingly, the two CaMBDs in Dictyostelium Cdk5 are strongly conserved in human Cdk5 indicating that the interaction between CaM and Cdk5 in human cells warrants investigation (Table 2). In Dictyostelium, Cdk5 function has been linked to endocytosis, phagocytosis, secretion, cell proliferation, cell differentiation, and multicellular development [10] [11] [12]. The protein has also been characterized as a nucleocytoplasmic protein that exits the nucleus during mitosis, only to return to the nucleus during cytokinesis [13]. "
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    ABSTRACT: 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.
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    • "The observations described in this study fit with those previously described by Sharma et al. [2002] who reported a 24-h delay in fruiting body formation for cells overexpressing a dominant negative form of Cdk5. Also, the number of fruiting bodies and spores that did form was significantly reduced compared to parental cells [Sharma et al., 2002]. These similarities, in addition to the expression profile of Cdk5 during development, the function of Cdk5 during cell proliferation, and the ability of roscovitine to inhibit Cdk5 activity during axenic growth, suggest that roscovitine, at least in part, inhibits Cdk5 during development possibly during the mitotic events that occur during the later stages of development. "
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    • "Since previous work was unable to generate cdk5 knockouts , other approaches are required to define the functions of this kinase (Sharma et al. 2002). In this regard, a recent pharmacological approach using the Cdk inhibitor roscovitine further supports a role for Cdk5 during axenic growth (Huber and O'Day 2011b). "
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