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Background: Previously, we found that amplification of chromosome 17q24.1-24.2 is associated with lymph node metastasis, tumour size, and lymphovascular invasion in invasive ductal carcinoma. A gene within this amplicon, CACNG4, an L-type voltage-gated calcium channel gamma subunit, is elevated in breast cancers with poor prognosis. Calcium homeostasis is achieved by maintaining low intracellular calcium levels. Altering calcium influx/efflux mechanisms allows tumour cells to maintain homeostasis despite high serum calcium levels often associated with advanced cancer (hypercalcemia) and aberrant calcium signaling. Methods: In vitro 2-D and 3-D assays, and intracellular calcium influx assays were utilized to measure tumourigenic activity in response to altered CANCG4 levels and calcium channel blockers. A chick-CAM model and mouse model for metastasis confirmed these results in vivo. Findings: CACNG4 alters cell motility in vitro, induces malignant transformation in 3-dimensional culture, and increases lung-specific metastasis in vivo. CACNG4 functions by closing the channel pore, inhibiting calcium influx, and altering calcium signaling events involving key survival and metastatic pathway genes (AKT2, HDAC3, RASA1 and PKCζ). Interpretation: CACNG4 may promote homeostasis, thus increasing the survival and metastatic ability of tumour cells in breast cancer. Our findings suggest an underlying pathway for tumour growth and dissemination regulated by CACNG4 that is significant with respect to developing treatments that target these channels in tumours with aberrant calcium signaling. Funding: Canadian Breast Cancer Foundation, Ontario; Canadian Institutes of Health Research. Keywords: Breast cancer metastasis, Gamma subunits, L-type channels, Voltage-gated calcium channels (VGCCs)

Copyright © by The American Society for Biochemistry and Molecular Biology, Mammalian AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that acts as a sensor of cellular energy status. AMPK is a heterotrimer of three different subunits, i.e. α, β and γ, with α being the catalytic subunit and β and γ having regulatory roles. Although several studies have defined different domains in α and β involved in the interaction with the other subunits of the complex, little is known about the regions of the γ subunits involved in these interactions. To study this, we have made sequential deletions from the N-termini of the γ subunit isoforms and studied the interactions with α and β subunits, both by two hybrid analysis and by co-immunoprecipitation. Our results suggest that a conserved region of 20-25 amino acids in γ1, γ2 and γ3, immediately N-terminal to the Bateman domains, is required for the formation of a functional, active αβγ complex. This region is required for the interaction with the β subunits. The interaction between the α and γ subunits does not require this region and occurs instead within the Bateman domains of the γ subunit, although the α-γ interaction does appear to stabilize the β-γ interaction. In addition, sequential deletions from the C-termini of the γ subunits indicate that deletion of any of the CBS motifs prevents the formation of a functional complex with the α and β subunits., This study was supported by the EXGENESIS Integrated Project (LSHM-CT-2004-005272) funded by the European Commission. Studies in the PS laboratory were also supported by the Spanish Ministry of Education and Science grant SAF2005-00852. Studies in the DGH laboratory were also supported by a Programme Grant from the Wellcome Trust.

In der vorliegenden Arbeit wurden erstmals drei neue modulatorische Gamma-Untereinheiten (Gamma 3-5) spannungsabhängiger Kalziumkanäle kloniert, ihre Gewebeverteilung durch Northern blot-Analysen und In situ-Hybridisierungen sowie ihre Funktion anhand von Koexpressionsstudien bestimmt. Die neuronalen Untereinheiten (Gamma 2-4) führen bei Koexpression mit L- und P/Q-Typ Untereinheiten zu einer Verschiebung der Steady state-Inaktivierungskurve in Richtung negativerer Potentiale. Die ubiquitäre Gamma 5 Untereinheit beschleunigt die Aktivierung und Inaktivierung von T-Typ Untereinheiten über den gesamten Potentialbereich. Dies ist der erste Hinweis darauf, daß auch T-Typ Kanäle ähnlich wie L-Typ Kanäle aus mehreren Untereinheiten aufgebaut sein könnten. In the present work we cloned for the first time three novel auxiliary gamma subunits (gamma 3-5) of voltage gated calcium channels. Their distribution in different tissues was assessed by northern blot and in situ hybridisation-techniques. Coexpression studies served to functionally characterize the electrophysiological properties of the newly identified subunits. Upon coexpression with L- and P/Q-type calcium channel subunits the neuronal gamma 2-4 shift the steady state-incativation curve towards more hyperpolarized potentials. The ubiquitous gamma 5 increases the timecourse of activation and inactivation when coexpressed with T-type channels. This is the the first evidence reported that T-type calcium channels - like their L-type counterparts - may consist of different subunits.