THE NETWORK ORGANIZATION AND THE PHOSPHORYLATION OF CYTOKERATINS ARE CONCOMITANTLY MODIFIED BY FORSKOLIN IN THE ENTEROCYTE-LIKE DIFFERENTIATED CACO-2 CELL-LINE

Confluent Caco-2 cells, originating from a human colon carcinoma, disp lay morphological and functional characteristics of differentiated ent erocytes such as the presence of a polarized monolayer covered by an a pical brush border that express several hydrolases. The adaptation of these cells to grow in the continuous presence of forskolin, a drug kn own to stimulate adenylyl cyclase permanently, has been previously sho wn to result in a decreased apical expression of hydrolases and in mor phological alterations including the disappearance of intercellular sp aces and shortening of microvilli. In the present work we have analyze d the possibility that cytoskeletal proteins may be the target of fors kolin in living Caco-2 cells. We show that forskolin initiates dramati c changes in the spatial organization of the cytokeratin network that correlate with an increased phosphorylation of cytokeratin molecules, whereas microtubules, microfilaments and vimentin remain mainly unaffe cted. Indirect immunofluorescence studies show that the cytokeratin ne twork is redistributed from the cell periphery to the cytoplasm. Bioch emical experiments indicate that forskolin doesn't interfere with the cytokeratin profile, since the three cytokeratins normally found in in testine (CK 8, CK 18, CK 19) are similarly expressed in both control a nd forskolin-Caco-2 cells. Analysis of P-32-labeled cytokeratin extrac ted from the two cell populations demonstrates that forskolin quantita tively increases the phosphorylation of type I cytokeratin (CK 18 and CK 19), whereas the phosphorylation of type II cytokeratin (CK 8) is a ltered both quantitatively and qualitatively with the emergence of a n ew phosphorylation site. These results provide a new cell system in-wh ich it is possible to control the subcelluar distribution of cytokerat in by changing their phosphorylation status and therefore to study the ir potential cellular functions.