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Nucleocytoplasmic transfer of cyclin dependent kinase 5 and its binding to puromycin-sensitive aminopeptidase in Dictyostelium discoideum

Abstract

The Dictyostelium discoideum homolog of mammalian cyclin dependent kinase 5 (Cdk5) has previously been shown to be required for optimal growth and differentiation in this model organism, however, the subcellular localization of the protein has not previously been studied. In this study, immunolocalizations and a GFP fusion construct localized Cdk5 predominantly to the nucleus of vegetative cells. Western blots showed that Cdk5 was present in both nuclear and non-nuclear fractions, suggesting a functional role in both cellular locales. During the early stages of mitosis, Cdk5 gradually moved from a punctate nucleoplasmic distribution to localize adjacent to the inner nuclear envelope. During anaphase and telophase, Cdk5 localized to the cytoplasm and was not detected in the nucleoplasm. Cdk5 returned to the nucleus during cytokinesis. Proteolytic activity has been shown to be a critical regulator of the cell cycle. Immunoprecipitations coupled with immunolocalizations identified puromycin-sensitive aminopeptidase A (PsaA) as a potential Cdk5 binding partner in Dictyostelium. Immunoprecipitations also identified two phosphotyrosine proteins (35 and 18 kDa) that may interact with Cdk5 in vivo. Together, this work provides new insight into the localization of Cdk5, its function during cell division, and its binding to a proteolytic enzyme in Dictyostelium.

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Abbreviations

Cdk:

Cyclin dependent kinase

Crp:

Cdc2-related PCTAIRE

Psa:

Puromycin sensitive aminopeptidase

KLH:

Keyhole limpet hemocyanin

IP:

Immunoprecipitation

GFP:

Green fluorescent protein

NumA:

Nucleomorphin A

Cbp4a:

Calcium-binding protein 4a

BSA:

Bovine serum albumin

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

kDa:

Kilodalton

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Acknowledgments

The authors would like to thank Dr. Yali Wang (Advanced Syntech Corporation, Mississauga, ON, Canada) for providing the peptides for antibody production and Andres Suarez for help in generating the AX3/[act15]:cdk5:GFP strain. This work was supported by a grant (DHO’D; A6807) and Canada Graduate Scholarship (RJH) from the Natural Sciences and Engineering Research Council of Canada and an Ontario Graduate Scholarship (RJH).

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Correspondence to Danton H. O’Day.

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Huber, R.J., O’Day, D.H. Nucleocytoplasmic transfer of cyclin dependent kinase 5 and its binding to puromycin-sensitive aminopeptidase in Dictyostelium discoideum . Histochem Cell Biol 136, 177 (2011). https://doi.org/10.1007/s00418-011-0839-6

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  • DOI: https://doi.org/10.1007/s00418-011-0839-6

Keywords

  • Cyclin dependent kinase 5
  • Puromycin sensitive aminopeptidase A
  • Nucleocytoplasmic localization
  • Mitosis
  • Dictyostelium discoiduem
  • Proteolysis