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Investigation of Phosphorylation Site Responsible for CaLP (P. fucata) Nucleo-cytoplasmic Shuttling Triggered by Overexpression of p21Cip1

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Abstract

Calmodulin (CaM) is a highly conserved and ubiquitous Ca2+-binding protein regulating intracellular Ca2+ concentration by acting as a sensor of this divalent cation in eukaryotic cells. Being such a very important signal sensor, CaM is susceptible to undergo many posttranslational modifications. One of these important modifications is its phosphorylation. Our previous investigations showed that CaM and calmodulin-like protein (CaLP) cloned from Pinctada fucata have many different characteristics in spite of their high similarity to each other. We have narrowed down that the C-terminal domains of CaM and CaLP are responsible for their discrepant subcellular localizations and shuttling of CaLP when it is co-transfected with p21Cip1, which is commonly considered as an important cell cycle regulating protein. In this study, we first predicted the potential phosphorylation site responsible for the shuttling and confirmed by fluorescence confocal microscopy. Together with fluorescence activated cell sorter analysis, we further investigated the releasing ability of wild type and point mutated CaLP from arrested cell cycle caused by p21Cip1 overexpression. By performing pull-down analysis and phosphorylation status of CaLP in cytoplasm fraction of transfected COS-7 cells with CaLP alone and phosphorylation status of CaLP in nuclear fraction of co-transfected COS-7 cells with CaLP and p21Cip, we propose that the CaLP staying in the cytoplasm is in the state of phosphorylation, but when p21Cip1 is overexpressed in mammalian cells, some signal triggers CaLP dephosphorylation and translocation into the nucleus.

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Acknowledgments

This work was financially supported by the National High Technology Research and Development Program of China (2006AA09Z413, 2006AA09Z441, 2006AA10A415) and the National Natural Science Foundation of China (no. 30530600).

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Authors and Affiliations

  1. Institute of Marine Biotechnology, Department of Biological Science and Biotechnology, Tsinghua University, Beijing, 100084, China

    Z. Fang, Q. Wang, W. Cao, Q. Feng, C. Li, L. Xie & R. Zhang

  2. Protein Science Laboratory of the Ministry of Education, Tsinghua University, Beijing, 100084, China

    L. Xie & R. Zhang

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  1. Z. Fang
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Correspondence to L. Xie.

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Fang, Z., Wang, Q., Cao, W. et al. Investigation of Phosphorylation Site Responsible for CaLP (P. fucata) Nucleo-cytoplasmic Shuttling Triggered by Overexpression of p21Cip1 . Mar Biotechnol 11, 270–279 (2009). https://doi.org/10.1007/s10126-008-9142-5

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