Phosphorylation of specific rat intestinal microvillus and basal-lateral membrane proteins by cyclic nucleotides

Abstract

The phosphorylation of specific membrane proteins by endogenous protein kinases in the microvillus and basal-lateral membrane components of rat intestinal epithelial cells has been studied using the technique of acrylamide slab-gel electrophoresis in the presence of sodium dodecyl sulfate. In the microvillus membrane, the endogenous phosphorylation of one specific membrane polypeptide, with a mol wt of 103,000, was found to be regulated by cyclic nucleotides. The rapid phosphorylation of this protein, which reached maximal levels at 1 min, was stimulated 2.2 times by 0.5 μM cyclic GMP, a concentration of the cyclic nucleotide that maximally activated the endogenous protein kinase. In comparison, the concentration of cyclic AMP required for maximal stimulation of phosphorylation was approximately 5 μM. A cyclic nucleotide-dependent phosphorylation of a specific membrane component was also observed in basal-lateral membranes. One of the major components of the basal-lateral membrane with a mol wt of 113,000 was found to be the specific phosphate acceptor for the endogenous, membrane-bound protein kinase. The phosphorylation of this polypeptide was maximally stimulated twofold by either 0.5 μM cyclic GMP or 5 μM cyclic AMP. These results suggest that in the small intestine, cyclic nucleotide-mediated effects on water and electrolyte transport may involve regulation of the level of phosphorylation of unique protein constituents of the basal-lateral and microvillus membrane.