Phosphorylation of keratin intermediate filaments (IF)is known to affect their assembly state and organization; however, little is known about the mechanisms regulating keratin phosphorylation. In this study, we demonstrate that shear stress, but not stretch, causes disassembly of keratin IF in lung alveolar epithelial cells (AEC) and that this disassembly is regulated by protein kinase C ␦-mediated phosphorylation of keratin 8 (K8) Ser-73. Specifically, in AEC subjected to shear stress, keratin IF are disassembled, as reflected by their increased solubility. In contrast, AEC subjected to stretch showed no changes in the state of assembly of IF. Pretreatment with the protein kinase C (PKC) inhibitor, bisindolymaleimide, prevents the increase in solubility of either K8 or its assembly partner K18 in shearstressed AEC. Phosphoserine-specific antibodies demonstrate that K8 Ser-73 is phosphorylated in a time-dependent manner in shear-stressed AEC. Furthermore, we showed that shear stress activates PKC ␦ and that the PKC ␦ peptide antagonist, ␦ V1-1, significantly attenuates the shear stress-induced increase in keratin phosphorylation and solubility. These data suggested that shear stress mediates the phosphorylation of serine residues in K8, leading to the disassembly of IF in alveolar epithelial cells. Importantly, these data provided clues regarding a molecular link between mechanically induced signal transduction and alterations in cytoskeletal IF.

We have isolated a novel, high M r protein from human retinal pigment epithelial cells and endothelial cells by affinity chromatography on Sepharose 4B. Two polypeptides are present on SDS-gels of the 8 M urea eluent with apparent molecular mass of ϳ210 and 47 kDa. In the absence of dithiothreitol, the two polypeptides migrate as one protein band with an apparent molecular mass of ϳ550 kDa. "Piglet," as this molecule is tentatively named, is present in retinal pigment epithelial and endothelial cells of several species, but could not be detected in the nonepithelial cells we examined. Immunofluorescent localization using an antibody to the 210-kDa polypeptide revealed a filamentous network in the cytoplasm of cultured cells. This antibody was used to identify a cDNA for piglet in a bovine aortic endothelial cell expression library. Sequence data indicate a high degree of identity with non-muscle myosin II heavy chain. We subsequently found that piglet had an actinactivated ATPase activity, colocalized with actin in cells, and reacted on Western blots with a pan-non-muscle myosin II heavy chain antiserum. The protein was also recognized by antibodies specific for myosin heavy chain isoform A, but did not react with anti-isoform B antibodies. Although piglet has several features in common with known forms of non-muscle myosin II, the distinctly unconventional features it displays suggest that it is a novel myosin.The retinal pigment epithelium (RPE) 1 is composed of a population of cuboidal to low columnar cells sandwiched between the neural retina and the highly vascularized choroid layer of the eye. Although RPE cells are normally nonproliferative and nonmigratory, they are highly active in other respects. They are indespensible for the function and survival of their immediate neighbors, the rods and cones, and malfunctions in the RPE form the basis for a variety of blinding disorders.The RPE cell feature that is perhaps most directly related to vision is the presence of dense pigment granules in their cytoplasm. These granules absorb light that has passed through the neural layers of the retina, preventing backscatter and unwanted reflection. The RPE also performs a variety of metabolic functions in support of the neural retina. By means of tight junctions, they create the blood-ocular barrier that regulates the passage of molecules between the retina and choroidal capillaries (1). They are, therefore, primary determinants of the photoreceptor microenvironment. The RPE is also responsible for phagocytosis and disposal of shed photoreceptor outer segments (2). In the absence of this function, degeneration of the neural retina ensues. Moreover, RPE are essential for production of the visual pigment rhodopsin (3). During the visual cycle, the 11-cis-retinaldehyde chromophore of rhodopsin present in photoreceptor cells is reduced to all-trans-retinol. To regenerate rhodopsin, the spent product is transferred to the RPE where it is isomerized to the 11-cis form once again. Directly and indirectly, therefore, the R...