Altered type I protein kinase in adhesion defective CHO cell variants

AD/sup v/ cells are Chinese hamster ovary (CHO) cell variants which cannot adhere to fibronectin coated substrata. The authors have shown that the defect in some clones of AD/sup v/ cells is distal to the initial interaction between fibronectin and its cell surface receptors, and that it extends to fibronectin medicated aggregation and endocytosis. The adhesion defect in some AD/sup v/ clones can be corrected by raising intracellular cAMP levels. Here they examine the protein kinase activities and phosphorylation patterns in an adhesion defective variant clone AD/sup v/F11CA11. Analysis of the cAMP dependent protein kinase activity (cAdPK) in crude extracts of F11CA11 cells shows an apparent increase in K (activation) as compared to wild type (WT) CHO cell extracts. Binding studies with 3H-cAMP reveal that the type I peak in F11CA11 has a K/sub d/ of 1.7 x 1008 M as compared to 2.0 x 1009 M for WT, whereas the type II peak K/sub d/ is approximately 1 x 10 09 M for both WT and F11CA11. Two-dimensional polyacrylamide gel analysis of 32Pi labeled WT cells and F11CA11 cells with or without cAMP treatment reveals the presence of a protein(s) of 50 kilodaltons which is phosphorylated in WT cells and in cAMP treated F11CA11 cells but not in untreated F11CA11 cells. These findings, coupled with previous observations, strongly indicate that the adhesion defect in AD/sup v/F11CA11 cells is associated with an altered type I cAdPK having lower affinity for cAMP. At normal cellular cAMP levels this enzyme fails to phosphorylate one or more critical protein substrates; however, by raising internal cAMP levels, the defect can be overcome