Cytosolic phosphorylation of calnexin controls intracellular Ca2+ oscillations via an interaction with SERCA2b

Calreticulin (CRT) and calnexin (CLNX) are lectin chaperones that participa te in protein folding in the endoplasmic reticulum (ER). CRT is a soluble E R lumenal protein, whereas CLNX is a transmembrane protein with a cytosolic domain that contains two consensus motifs for protein kinase (PK) C/prolin e-directed kinase (PDK) phosphorylation, Using confocal Ca2+ imaging in Xen opus oocytes, we report here that coexpression of CLNX with sarco endoplasm ic reticulum calcium ATPase (SERCA) 2b results in inhibition of intracellul ar Ca2+ oscillations, suggesting a functional inhibition of the pump. By si te-directed mutagenesis, we demonstrate that this interaction is regulated by a COOH-terminal serine residue (S562) in CLNX. Furthermore, inositol 1,4 ,5-trisphosphate-mediated Ca2+ release results in a dephosphorylation of th is residue. We also demonstrate by coimmunoprecipitation that CLNX physical ly interacts with the COOH terminus of SERCA2b and that after dephosphoryla tion treatment, this interaction is significantly reduced. Together, our re sults suggest that CRT is uniquely regulated by ER lumenal conditions, wher eas CLNX is, in addition, regulated by the phosphorylation status of its cy tosolic domain. The S562 residue in CLNX acts as a molecular switch that re gulates the interaction of the chaperone with SERCA2b, thereby affecting Ca 2+ signaling and controlling Ca2+-sensitive chaperone functions in the ER.