Study On The Distribution And Function Of CaMK Ⅱ In HeLa Cell Cycle Regulation

Ca2+/CaM-dependent protein kinase II (CaMK II) is a ubiquitously expressed protein kinase that transduce elevated Ca2+ signals in cells to a number of target proteins ranging from ion channels to the tran-scriptional activators.CaMK II have a unique holoenzyme structure and autoregulatory property that allow it to give a prolonged response totransient Ca2+ signals and to sense cellular Ca2+ oscillations.The mul-tifunction of CaMK II in calcium signaling strongly suggests that it play an important role in cell cycle regulation.It has been well known that protein's intracellular distribution is correlative with its functions.In this article, we first observed CaMK II 's distribution in living HeLa cell using GFP fusion gene tech-nique.We also examined its functions in the regulation of mitosis byboth inhibiting the endogenous CaMK II "s activity and overexpressing the CaMK II in HeLa cells.Followed are our main results:1.Study of the distribution of CaMK II in HeLa cells as revealed by GFP fusion gene technique.We constructed CaMKIIp-GFP fusion gene andthe expression of fusion protein was confirmed by Western blotting assay.Fluorescent microscope observation showed that CaMK IIP-GFP fusion protein mainly localized in interphase nucleus and in the whole cell during mitotic phase,the same pattern as endogenous CaMK II P as showed by indirect immunostaining.These result showed the precisenessof GFP fusion protein technique in the study of intracellular distri-bution of target protein.2.Inhibition of endogenous CaMK II 's activity resulted in the delay of M/A transition while overexpression of CaMK II p-GFP promoted G2/M and M/A transitions,indicating that CaMK II may positively regulate the G2/M and M/A transition.3.Overexpression of GFP-CaMKIIct caused a delay in G2/M transition and Metaphas to anaphase transition.(1)We first constructed the cons-titutively activemutant of CaMK II by truncation at autoinhibiting region and constructed the GFP-CaMK II ct fusion gene by put the mutantat the C-terminal of GFP gene. Expression of GFP-CaMK II ct fusion prot-ein was confirmed by Western blotting assay and fluorescent microscope observation showed that GFP-CaMK II ct fusion protein mainly localized in interphase cytoplasm and in the whole cell of mitotic phase, different from the wildtype CaMK II .This result showed that the C-terminal region of CaMK II is necessary for its entry into nucleus.Overexpression of GFP-CaMK II ct in HeLa cells caused delay in G2/M and M/A transitions, indicating that the constitutive kinase activity, different from the specific activation of endogenous CaMK II .may cause unnecessary phosphorylation of its substrates at transitions and disturb the normal signaling pathways, resulted in the delay of G2/M and metaphse to anaphase transitions.Taken together,our results indicated that functions of CaMK II in mitosis is precisely regulated by calcium signal in HeLa cells and the timing and distribution of CaMK II are of great importance to its positive role in regulation of mitosis.4.For further study of the molecular mechanism of CaMK II 's function in regulation of mitosis, we established Tet-off HeLa cell line by transfecting HeLa cells with pTet-off plasmid and screened with G418.Among the cell clones obtained, clone NO.7 showed a low background and high inducibility.We also constructed the pTRE-CaMK II p-GFP and pTRE-CaMK II ct vectors and prepared for the establishment of double stable CaMK II inducible expression HeLa cell line.