Phosphorylation of cytoskeletal proteins tightly associated with the vascular smooth muscle membranes.

Abstract

A study was performed to determine whether the proteins phosphorylated by cAMP dependent and calmodulin dependent protein kinase in vascular smooth muscle membrane fractions represent integral membrane proteins or are tightly associated cytoskeletal proteins. In the unextracted microsomal fraction cAMP dependent protein kinase phosphorylated proteins of 240K, 105K, and 48K daltons. Similarly, calmodulin dependent protein kinase phosphorylated 65K, 60K, 48K, and 20K dalton bands. The 48K dalton band represented a major protein in the microsomal fraction, and it was a common substrate for both cAMP dependent and calmodulin dependent protein kinase, and the extent of phosphorylation by two kinases was additive. The 48K dalton band showed immunological reaction with monoclonal antibodies raised against human umbilical artery F actin, and it also comigrated with arterial smooth muscle actin on SDS gel electrophoresis. Plasma membranes prepared from vascular smooth muscle microsomes after extraction with actomyosin extraction buffer consisting of 2 mmol X litre-1 TRIS-maleate, pH 6.8, 0.25 mol X litre-1 sucrose, 0.5 mmol X litre-1 ATP, 0.2 mmol X litre-1 CaCl2, 0.1 mmol X litre-1 phenylmethylsulphonylfluoride, and 1 mmol X litre-1 dithiothreitol yielded membranes that were substantially free from cytoskeletal protein contamination. These membranes were enriched 60-80 fold in plasma membrane marker enzymes and showed energy dependent calcium uptake. In the extracted plasma membranes none of the proteins was phosphorylated by cAMP dependent or calmodulin dependent protein kinase. Thus these results show that protein phosphorylated in the unextracted microsomal fraction or unextracted plasma membranes are not integral plasma membrane proteins but represent tightly associated cytoskeletal proteins.