CaMKK2 facilitates Golgi-associated vesicle trafficking to sustain cancer cell proliferation

CaMKK2 (Calcium/Calmodulin Dependent Protein Kinase Kinase 2) is an important component of multiple signalling pathways that regulate glucose metabolism, adipogenesis, nutrient intake and has been shown to be deregulated in diseases such as obesity and cancer. We first identified CAMKK2 as an androgen receptor-regulated kinase that sustains prostate cancer cell metabolism and tumorigenesis. An important function of CaMKK2 is to phosphorylate and activate AMPK (AMP-activated protein kinase), a central metabolic rheostat in all cell types that can regulates the balance between anabolic and catabolic metabolism by regulating the activity of metabolic enzymes and autophagy. Importantly, it was recently shown that the growth inhibitory effects of CAMKK2 knockout in prostate cancer cells cannot be rescued through the use of AMPK activators suggesting that this kinases affects cell proliferation through other mechanisms too. The aim of our study was to provide insights into the impact of CaMKK2 on cell proliferation by identifying and functionally characterising novel interactions partners using proteomics, imaging and in vitro assays. Our proteomics study has identified many novel CaMKK2 interactors involved in membrane trafficking and RNA processing. We have mapped a binding peptide capable of interacting with CaMKK2 in one of these proteins, Gemin4, and have shown that this also enriches COPI coatomer subunits. COPI-coated vesicles facilitate retrograde membrane trafficking from the Golgi to the endoplasmic reticulum. This led us to hypothesise that perturbing CaMKK2 expression might impact on organelles within the secretory pathway. Imaging experiments of CaMKK2 knockdown LNCaP cells showed an expanded Golgi, which aligned with an observation that CaMKK2 regulated both Gemin4 and COPI coatomer subunit stability. Furthermore, inhibition of CaMKK2 kinase activity or knockdown of Gemin4 reduced COPI coatomer protein expression. Gemin4 exists in a number of multiprotein complexes together with Gemin1-6 and is known to interact with alpha- and delta-COP. COPI coat components have been reported to be critical for cell survival due to their role in lysosomal maturation and mitochondrial metabolism. The CaMKK2 knockdown cells showed a significant reduction in lysosomal acidification and increased sensitivity to inhibitors of the vacuolar proton ATPase. Based on our findings we suggest that CAMKK2 sustains cell proliferation in large part through effects on organelle integrity and COPI-mediated membrane trafficking. These results indicate for the first time that CaMKK2 expression and activity affects COPI vesicle trafficking.