Investigation of phosphorylation site responsible for CaLP (P. fucata) nucleo-cytoplasmic shuttling triggered by overexpression of p21Cip1.
Calmodulin (CaM) is a highly conserved and ubiquitous Ca(2+)-binding protein regulating intracellular Ca(2+) 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 p21(Cip1), 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 p21(Cip1) 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 p21(Cip), we propose that the CaLP staying in the cytoplasm is in the state of phosphorylation, but when p21(Cip1) is overexpressed in mammalian cells, some signal triggers CaLP dephosphorylation and translocation into the nucleus.
Fang
Z
Z
Institute of Marine Biotechnology, Department of Biological Science and Biotechnology, Tsinghua University, Beijing, 100084, China.
Wang
Q
Q
Cao
W
W
Feng
Q
Q
Li
C
C
Xie
L
L
Zhang
R
R
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism*; D002522:Chlorocebus aethiops; D015870:Gene Expression; D010766:Phosphorylation; D049911:Pinctada / Q000235:genetics* / Q000378:metabolism*; D021381:Protein Transport
D002147:Calmodulin