Associations of PKC Isoforms with the Cytoskeleton of B16F10 Melanoma Cells

Abstract

Although PKC plays a major role in regulating the morphology and function of the cytoskeleton, little is known about in situ associations of specific isoforms with the cytoskeleton. We demonstrate that seven PKC isoforms are expressed in B16F10 melanoma cells and show different levels of induction by serum. Using cell cytoskeleton preparations (CSKs), confocal microscopy, and immunocytochemistry, all isoforms show specific patterns of localization to focal contact-like structures (alpha, delta), very small cytoplasmic granules/vesicles (all isoforms), dense ordered arrays of small granules in the perinuclear region (alpha, delta), granules/vesicles associated with a homogeneous framework in the cytoplasm adjacent to the nucleus (gamma), or irregular-shaped patches of granules at or near the nuclear perimeter (eta, theta). In addition, several isoforms are present as cytoplasmic granules/ vesicles in linear or curvilinear arrays (alpha, delta, epsilon, theta). When isoform localization is examined using 3.7% formaldehyde or methanol:acetone, the patterns of localization in CSKs are often difficult or impossible to detect, and many are described here for the first time. Double-labeling experiments with CSK demonstrate that PKC actin co-localizes with punctate alpha-rich particles above the nucleus, granules of epsilon throughout the cytoplasm, and with theta in irregular-shaped aggregates associated with the nucleus. Vimentin co-localizes with perinuclear granules of delta and beta(2), and alpha-tubulin co-localizes with theta in structures at or near the nuclear surface and in microtubules associated with the microtubule organizing center (MTOC). In summary, the present study demonstrates that seven PKC isoforms are endogenously expressed in B16F10 melanoma cells. These isoforms show various levels of induction by serum and specific patterns of association with various components of the detergent-resistant cell cytoskeleton.

Tumor suppressor RASSF1A is a microtubule-binding protein that stabilizes microtubules and induces G2/M arrest

“…Immunostaining to detect a-tubulin was performed as previously described with modifications (Szalay et al, 2001). Briefly, cells growing on Lab-tek slides were transiently transfected with expression vector carrying GFP cDNA or GFP-RASSF1A fusion construct.…”

Wnt11 Signaling Promotes Proliferation, Transformation, and Migration of IEC6 Intestinal Epithelial Cells

“…types (64,65). To determine whether redistribution of E-cadherin and the apparent loss of E-cadherin-mediated cell-cell contacts affected cell movement or migration, we performed a scratch assay (Fig.…”

Insulin Receptor-Protein Kinase C-γ Signaling Mediates Inhibition of Hypoxia-Induced Necrosis of Cortical Neurons

“…In most cases, activated PKC isoforms are translocated to plasma membranes, but in some cases they are translocated to several cytosolic organelles (Shirai and Saito, 2002). There are reports that activated PKC-␥ is translocated to Golgi-like organelles (Goodnight et al, 1995) or a perinuclear region (Szalay et al, 2001) as well as to plasma membranes (Goodnight et al, 1995;Szalay et al, 2001), although the role of these translocations in cellular signaling remains to be determined. In the present study, insulin stimulation of cortical neurons also caused translocation of PKC-␥ to a perinuclear region, although it remains to be determined whether it is a direct consequence of insulin receptor activation.…”