ISSN:
0021-9541
Keywords:
Life and Medical Sciences
;
Cell & Developmental Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Medicine
Notes:
ADv cells are Chinese hamster ovary (CHO) cell variants which cannot adhere to fibronectin coated substrata (Harper amp; Juliano: J. Cell Biol., 1980; Nature 1981a, b). We have shown that the defect in some clones of ADv cells is distal to the initial interaction between fibronectin and its cell surface receptors (Cheung and Juliano: Exp. Cell Res., 1984), and that it extends to fibronectin mediated aggregation and endocytosis. The adhesion defect in some ADv clones can be corrected by raising intracellular cAMP levels (Cheung & Juliano: J. Cell Physiol., 1985). Here we examine the protein kinase activities and phosphorylation patterns in an adhesion defective variant clone ADv 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. Further, the DE-52 cellulose chromatography profile of cAdPK in the F11CA11 variant is markedly different from WT in that the type I cAdPK peak elicited by 1 μM cAMP is essentially missing in F11CA11, while the type II cAdPK peak is similar to that in WT. Raising the cAMP level to 100 μM elicits a type I peak in F11CA11 with about 45% of the activity of the WT peak. Binding studies with 3H-cAMP reveal that the type I peak in F11CA11 has a Kd of 1.7 times; 10-8 M as compared to 2.0 × 10-9 M for WT, whereas the type II peak Kd is approximately 1 × 10-9 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 our previous observations, strongly indicate taht the adhesion defect in ADvF11CA11 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. Thus type I cAdPK seems to play an important role in the regulation of fibronectin mediated cell adhesion, cell aggregation, and endocytosis.
Additional Material:
6 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/jcp.1041300117
Permalink
