Calcium sensing proteins regulate signalling pathways in response to changes in intracellular calcium levels. The prototypical calcium sensor, Calmodulin, can modulate kinase activity, notably those of the Ca2+-Calmodulin-dependent protein kinase (CAMK) family. Another group of Ca2+-sensors are the CREC proteins, which include Calumenin, Reticulocalbin 1, Reticulocalbin 3, ERC55 (Reticulocalbin 2), and Cab45. In contrast to Calmodulin, there is a massive blind spot in the knowledge concerning the function of the CREC family, structure, and their downstream effector proteins (306 PubMed entries for the entire CREC family compared to >47,000 for Calmodulin).
We recently reported for the first time that CREC proteins can regulate protein kinases, where we described allosteric inhibition of the catalytic activity of PSKH1 by Reticulocalbin-3. To expand on this discovery, we have undertaken a proteome-wide screen to map the interactomes of CREC family members, with a focus on determining how broadly CREC proteins act in protein kinase regulation. To this end, we undertook two complementary proteomics studies to define the CREC proteins’ interactomes. We used purified biotinylated CREC proteins for Streptavidin pulldowns in the presence and absence of calcium using lysates of two distinct cell lines. We complemented pulldown studies with proximity labelling experiments in cells using TurboID fusions for each CREC protein. We subsequently validated CREC interactions with a subset of kinase interactors using recombinant proteins, revealing the breadth and selectivity of mechanisms by which CREC proteins can modulate kinase activity. Our findings advance fundamental knowledge of the CREC family and illuminate our understanding of unconventional modes of regulation by Ca2+-sensors. Overall, this study serves as an invaluable resource in this emerging field of study.