Phosphorylation by CK2 and MAPK enhances calnexin association with ribosomes

Calnexin was initially identified as an endoplasmic reticulum (ER) type I i ntegral membrane protein, phosphorylated on its cytosolic domain by ER-asso ciated protein kinases, Although the role of the ER luminal domain of calne xin has been established as a constituent of the molecular chaperone machin ery of the ER, less is known about the role of the cytosolic phosphorylatio n of calnexin. Analysis by two-dimensional phosphopeptide maps revealed tha t calnexin was in vitro phosphorylated in isolated microsomes by casein kin ase 2 (CK2) and extracellular-signal regulated kinase-1 (ERK-1) at sites co rresponding to those for in vivo phosphorylation, In canine pancreatic micr osomes, synergistic phosphorylation by CK2 and ERK-1 led to increased assoc iation of calnexin with membrane-bound ribosomes. In vivo, calnexin-associa ted ERK-1 activity was identified by co-immunoprecipitation. This activity was abolished in cells expressing a dominant-negative MEK-1. Activation of ERK-1 in cells by addition of serum led to a 4-fold increase in ribosome-as sociated calnexin over unstimulated cells. Taken together with studies reve aling calnexin association with CK2 and ERK-1, a model is proposed whereby phosphorylation of calnexin leads to a potential increase in glycoprotein f olding close to the translocon.