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Phosphorylation of calcium/calmodulin-stimulated protein kinase II at T286 enhances invasion and migration of human breast cancer cells

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ABSTRACT

Calcium/calmodulin-stimulated protein kinase II (CaMKII) is a multi-functional kinase that controls a range of cellular functions, including proliferation, differentiation and apoptosis. The biological properties of CaMKII are regulated by multi-site phosphorylation. However, the role that CaMKII phosphorylation plays in cancer cell metastasis has not been examined. We demonstrate herein that CaMKII expression and phosphorylation at T286 is increased in breast cancer when compared to normal breast tissue, and that increased CAMK2 mRNA is associated with poor breast cancer patient prognosis (worse overall and distant metastasis free survival). Additionally, we show that overexpression of WT, T286D and T286V forms of CaMKII in MDA-MB-231 and MCF-7 breast cancer cells increases invasion, migration and anchorage independent growth, and that overexpression of the T286D phosphomimic leads to a further increase in the invasive, migratory and anchorage independent growth capacity of these cells. Pharmacological inhibition of CaMKII decreases MDA-MB-231 migration and invasion. Furthermore, we demonstrate that overexpression of T286D, but not WT or T286V-CaMKII, leads to phosphorylation of FAK, STAT5a, and Akt. These results demonstrate a novel function for phosphorylation of CaMKII at T286 in the control of breast cancer metastasis, offering a promising target for the development of therapeutics to prevent breast cancer metastasis.

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CaMKII overexpression alters the phosphorylation and expression of multiple proteins in breast cancer cells.(A) Expression and phosphorylation of 44 proteins were examined by Proteome Profile Human Phospho-Kinase Array following inducible overexpression of empty vector (EV), wild-type (WT), T286D phosphomimic mutant, or T286V phospho mutant forms of CaMKII in MDA-MB-231 cells. (B) The relative expression and phosphorylation of these proteins were quantitated by densitometric analysis, and expression normalised to that observed in the EV control cells. (C) The expression and phosphorylation of 7 proteins found to be differentially expressed/phosphorylated following T286D phosphomimic mutant overexpression in the array were confirmed by Western blot in MDA-MB-231 and MCF-7 cells. Blots are representative of three independent experiments. The relative expression of these proteins were quantitated by densitometric analysis, and expression normalised to that observed in EV (D) MDA-MB-231 and (E) MCF-7 cells. *denotes statistical significance from EV cells, Φdenotes statistical significance from WT cells, φdenotes significance from T286V expressing cells.
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f8: CaMKII overexpression alters the phosphorylation and expression of multiple proteins in breast cancer cells.(A) Expression and phosphorylation of 44 proteins were examined by Proteome Profile Human Phospho-Kinase Array following inducible overexpression of empty vector (EV), wild-type (WT), T286D phosphomimic mutant, or T286V phospho mutant forms of CaMKII in MDA-MB-231 cells. (B) The relative expression and phosphorylation of these proteins were quantitated by densitometric analysis, and expression normalised to that observed in the EV control cells. (C) The expression and phosphorylation of 7 proteins found to be differentially expressed/phosphorylated following T286D phosphomimic mutant overexpression in the array were confirmed by Western blot in MDA-MB-231 and MCF-7 cells. Blots are representative of three independent experiments. The relative expression of these proteins were quantitated by densitometric analysis, and expression normalised to that observed in EV (D) MDA-MB-231 and (E) MCF-7 cells. *denotes statistical significance from EV cells, Φdenotes statistical significance from WT cells, φdenotes significance from T286V expressing cells.

Mentions: All CaMKII overexpressing breast cancer cells had increased levels of pERK1/2 and vimentin and decreased levels of E-cadherin (Fig. 8A–E), and MDA-MB-231 cells also possessed elevated levels of pFAK (Fig. 8C,D). Furthermore, MDA-MB-231 (Fig. 8C,D) and MCF-7 cells (Fig. 8C,E) overexpressing the T286D phosphomimic mutant form of CaMKII had significantly elevated levels of pFAK, pSTAT5a and pAkt, when compared to EV, WT and T286V phospho control cells. This indicates that T286 phosphorylation of CaMKII can lead to increased activation of FAK, STAT5a and Akt.


Phosphorylation of calcium/calmodulin-stimulated protein kinase II at T286 enhances invasion and migration of human breast cancer cells
CaMKII overexpression alters the phosphorylation and expression of multiple proteins in breast cancer cells.(A) Expression and phosphorylation of 44 proteins were examined by Proteome Profile Human Phospho-Kinase Array following inducible overexpression of empty vector (EV), wild-type (WT), T286D phosphomimic mutant, or T286V phospho mutant forms of CaMKII in MDA-MB-231 cells. (B) The relative expression and phosphorylation of these proteins were quantitated by densitometric analysis, and expression normalised to that observed in the EV control cells. (C) The expression and phosphorylation of 7 proteins found to be differentially expressed/phosphorylated following T286D phosphomimic mutant overexpression in the array were confirmed by Western blot in MDA-MB-231 and MCF-7 cells. Blots are representative of three independent experiments. The relative expression of these proteins were quantitated by densitometric analysis, and expression normalised to that observed in EV (D) MDA-MB-231 and (E) MCF-7 cells. *denotes statistical significance from EV cells, Φdenotes statistical significance from WT cells, φdenotes significance from T286V expressing cells.
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f8: CaMKII overexpression alters the phosphorylation and expression of multiple proteins in breast cancer cells.(A) Expression and phosphorylation of 44 proteins were examined by Proteome Profile Human Phospho-Kinase Array following inducible overexpression of empty vector (EV), wild-type (WT), T286D phosphomimic mutant, or T286V phospho mutant forms of CaMKII in MDA-MB-231 cells. (B) The relative expression and phosphorylation of these proteins were quantitated by densitometric analysis, and expression normalised to that observed in the EV control cells. (C) The expression and phosphorylation of 7 proteins found to be differentially expressed/phosphorylated following T286D phosphomimic mutant overexpression in the array were confirmed by Western blot in MDA-MB-231 and MCF-7 cells. Blots are representative of three independent experiments. The relative expression of these proteins were quantitated by densitometric analysis, and expression normalised to that observed in EV (D) MDA-MB-231 and (E) MCF-7 cells. *denotes statistical significance from EV cells, Φdenotes statistical significance from WT cells, φdenotes significance from T286V expressing cells.
Mentions: All CaMKII overexpressing breast cancer cells had increased levels of pERK1/2 and vimentin and decreased levels of E-cadherin (Fig. 8A–E), and MDA-MB-231 cells also possessed elevated levels of pFAK (Fig. 8C,D). Furthermore, MDA-MB-231 (Fig. 8C,D) and MCF-7 cells (Fig. 8C,E) overexpressing the T286D phosphomimic mutant form of CaMKII had significantly elevated levels of pFAK, pSTAT5a and pAkt, when compared to EV, WT and T286V phospho control cells. This indicates that T286 phosphorylation of CaMKII can lead to increased activation of FAK, STAT5a and Akt.

View Article: PubMed Central - PubMed

ABSTRACT

Calcium/calmodulin-stimulated protein kinase II (CaMKII) is a multi-functional kinase that controls a range of cellular functions, including proliferation, differentiation and apoptosis. The biological properties of CaMKII are regulated by multi-site phosphorylation. However, the role that CaMKII phosphorylation plays in cancer cell metastasis has not been examined. We demonstrate herein that CaMKII expression and phosphorylation at T286 is increased in breast cancer when compared to normal breast tissue, and that increased CAMK2 mRNA is associated with poor breast cancer patient prognosis (worse overall and distant metastasis free survival). Additionally, we show that overexpression of WT, T286D and T286V forms of CaMKII in MDA-MB-231 and MCF-7 breast cancer cells increases invasion, migration and anchorage independent growth, and that overexpression of the T286D phosphomimic leads to a further increase in the invasive, migratory and anchorage independent growth capacity of these cells. Pharmacological inhibition of CaMKII decreases MDA-MB-231 migration and invasion. Furthermore, we demonstrate that overexpression of T286D, but not WT or T286V-CaMKII, leads to phosphorylation of FAK, STAT5a, and Akt. These results demonstrate a novel function for phosphorylation of CaMKII at T286 in the control of breast cancer metastasis, offering a promising target for the development of therapeutics to prevent breast cancer metastasis.

No MeSH data available.


Related in: MedlinePlus