The importance of protein phosphatases in the maintenance of cytoskeletal structure is supported by the serious liver injury caused by microcystin-LR, a hepatotoxic inhibitor of type-1 and type-2A serine/threonine protein phosphatases. We used the microcystin-LR-induced cell injury as a model to study the roles of protein dephosphorylation in maintaining cytoskeletal structure and cellular interactions in primary rat hepatocyte cultures. Confocal microscopy revealed that the first visible effect of microcystin-LR is disruption of desmoplakin organization at the cell surface, indicating dissociation of desmosomes. This effect is followed by a dramatic reorganization of both the intermediate filament (keratins 8 and 18) and microfilament networks, resulting in a merged structure in which the intermediate filaments are organized around a condensed actin core. Keratin 8, keratin 18 and desmoplakin I/II are the major cytoskeleton-associated targets for microcystin-LR-induced phosphorylation. Hyperphosphorylation of keratin 8 and 18 is accompanied by an increased keratin solubility, which correlates with the observed morphological effects. Phosphopeptide mapping shows that four specific tryptic phosphopeptides are highly phosphorylated predominantly in the soluble pool of keratin 18, whereas keratin 8 shows no indications of such assembly state-specific sites. Phosphopeptide maps of keratins phosphorylated in vivo and in vitro indicate that Ca2+/calmodulin-dependent kinase may be involved in regulating the serine-specific phosphorylation of both keratin 8 and keratin 18, while cAMP-dependent protein kinase does not seem to play a major role in this context. Taken together, our results show that the interactions between keratin intermediate filaments and desmosomes as well as the assembly states of their main constituent proteins, are directly regulated by serine/threonine kinase/phosphatase equilibria.