Identification of Two Distinct Pathways of Protein Kinase Cα Down-regulation in Intestinal Epithelial Cells

Abstract

Signal transduction pathways are controlled by desensitization mechanisms, which can affect receptors and/or downstream signal transducers. It has long been recognized that members of the protein kinase C (PKC) family of signal transduction molecules undergo downregulation in response to activation. Previous reports have indicated that key steps in PKC␣ desensitization include caveolar internalization, priming site dephosphorylation, ubiquitination of the dephosphorylated protein, and degradation by the proteasome. In the current study, comparative analysis of PKC␣ processing induced by the PKC agonists phorbol 12-myristate 13-acetate and bryostatin 1 in IEC-18 rat intestinal epithelial cells demonstrates that: (a) at least two pathways of PKC␣ down-regulation can co-exist within cells, and (b) a single PKC agonist can activate both pathways at the same time. Using a combined biochemical and morphological approach, we identify a novel pathway of PKC␣ desensitization that involves ubiquitination of mature, fully phosphorylated activated enzyme at the plasma membrane and subsequent down-regulation by the proteasome. The phosphatase inhibitors okadaic acid and calyculin A accelerated PKC␣ down-regulation and inhibitors of vesicular trafficking did not prevent degradation of the protein, indicating that neither internalization nor priming site dephosphorylation are requisite intermediate steps in this ubiquitin/proteasome dependent pathway of PKC␣ down-regulation. Instead, caveolar trafficking and dephosphorylation are involved in a second, proteasomeindependent mechanism of PKC␣ desensitization in this system. Our findings highlight subcellular distribution and phosphorylation state as critical determinants of PKC␣ desensitization pathways.The PKC 1 family of phospholipid-dependent serine/threonine kinases plays a central role in signal transduction and has been implicated in regulation of a variety of fundamental cellular processes, including cell growth and cell cycle progression (1), differentiation (2), apoptosis (3), and survival (4). The tumor promoting properties of PKC agonists such as phorbol esters, together with evidence for changes in the expression and/or activity of PKC isozymes in a variety of malignancies, point to a role for altered PKC signaling in neoplastic transformation (e.g. Ref. 5). Strict control of PKC function is achieved by two coordinated mechanisms: consecutive phosphorylation on three "priming sites" (activation loop, turn motif, and hydrophobic motif), which is required for catalytic competence (6), and binding of second messengers (diacylglycerol and phosphatidylserine), which promotes membrane targeting required for conformational activation (7). Membrane compartmentalization of PKC is stabilized by binding of the enzyme to anchoring proteins that interact exclusively with the active kinase (8). As a consequence of such spatial control, PKC is sequestered in the vicinity of its substrates and performs its biological function(s) through substrate phosphorylation. The active species ...

Regulation of Protein Kinase C function by phosphorylation on conserved and non-conserved sites

“…It should be noted however that while dephosphorylation serves as an important switch to turn off the activity of these enzymes, dephosphorylation does not necessarily predispose the enzyme to degradation [79]. Consistent with this, it has been reported that downregulation of PKC can occur through a ubiquitin/proteasome pathway that does not involve dephosphorylation of the enzyme [82].…”

Heat Shock Proteins Regulate Activation-induced Proteasomal Degradation of the Mature Phosphorylated Form of Protein Kinase C

“…Immunoblot Analysis-Cells were lysed with 1% SDS, which extracts PKCs from the cytosol, membrane, and nonionic detergent-insoluble fractions, and immunoblot analysis of whole cell lysates was performed as we have described (28,62) using SuperSignal West Pico ECL (ThermoScientific). The apparent molecular weight of bands was determined based on electrophoretic migration relative to prestained standard proteins (Bio-Rad/ThermoScientific).…”

“…Immunofluorescence Analysis-Cells, grown on glass coverslips and treated as indicated, were fixed in 2% formaldehyde/ PBS for 15 min at room temperature and processed for immunofluorescence microscopy as we have described previously, using 0.2% saponin for permeabilization (28). Antibodies were rabbit anti-C-terminal PKC␣ (1:500) and TRITC-conjugated anti-rabbit secondary antibody (1:100).…”

“…Based on evidence that (a) binding of lipid coactivators can sensitize PKC to phosphatases (23), (b) loss of phosphate at a single priming site predisposes the other sites to dephosphorylation (5,24), (c) dephosphorylated PKC is highly unstable, and (d) the activated enzyme can be internalized, with dephosphorylation occurring on intracellular vesicles (4, 25-37), it has been proposed that agonist-induced down-regulation involves vesicle-mediated accumulation of the enzyme in a perinuclear compartment (38), followed by dephosphorylation of priming sites by a protein phosphatase 2A-like activity (27,28) and/or PHLPP1/2 (26). The fully dephosphorylated protein is sequestered in a neutral detergent-insoluble fraction, where it is refractory to rephosphorylation (26,27,32), and is eventually degraded via a dephosphorylation-dependent, ubiquitin-mediated proteasomal mechanism (11,14,36).…”

“…In IEC-18 intestinal epithelial cells, PKC agonists can induce both proteasomal and nonproteasomal degradation of the enzyme. Proteasomal degradation occurs primarily at the plasma membrane, whereas internalization targets the protein for endolysosomal processing (28,39). Dephosphorylation may not be a prerequisite for down-regulation, because phosphorylated PKC␣ is ubiquitinated, and it is the phosphorylated form that is detected in early and late endosomal compartments following internalization (28, 39 -41).…”