Novel Protein Kinase C-Mediated Control of Orai1 Function in Invasive Melanoma

Abstract

The incidence of malignant melanoma, a cancer of the melanocyte cell lineage, has nearly doubled in the past 20 years. Wnt5A, a key driver of melanoma invasiveness, induces Ca²⁺ signals. To understand how store-operated calcium entry (SOCE) contributes to Wnt5A-induced malignancy in melanoma models, we examined the expression and function of STIM1 and Orai1 in patient-derived malignant melanoma cells, previously characterized as either highly invasive (metastatic) or noninvasive. Using both fluorescence microscopy and electrophysiological approaches, we show that SOCE is greatly diminished in invasive melanoma compared to its level in noninvasive cell types. However, no loss of expression of any members of the STIM and Orai families was observed in invasive melanoma cells. Moreover, overexpressed wild-type STIM1 and Orai1 failed to restore SOCE in invasive melanoma cells, and we observed no defects in their localization before or after store depletion in any of the invasive cell lines. Importantly, however, we determined that SOCE was restored by inhibition of protein kinase C, a known downstream target of Wnt5A. Furthermore, coexpression of STIM1 with an Orai1 mutant insensitive to protein kinase C-mediated phosphorylation fully restored SOCE in invasive melanoma. These findings reveal a level of control for STIM/Orai function in invasive melanoma not previously reported.

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Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.01500-14.

ACKNOWLEDGMENTS

We thank Maya Shmurak and Michael O'Connell for technical support with this project. In addition, we thank Christoph Romanin (Johannes Kepler University, Linz, Austria) for providing us with the Orai1_Δ47 construct.

This work was supported by NIH grant 5R01GM097335 (J.S.) and R01HL097111 (M.T.).

R.H. performed and designed experiments, analyzed data, and contributed to the writing of the manuscript. X.Z., M.W., C.G., J.K., and K.M. performed experiments and analyzed data. M.T., D.L.G., and A.T.W. designed experiments and contributed to the writing of the manuscript. J.S. designed experiments, analyzed data, and wrote the manuscript.

We have no conflict of interest to declare.