Subcellular Translocation Research Articles

Phosphorylation and associated translocation of the 87-kDa protein, a major protein kinase C substrate, in isolated nerve terminals.

A protein of 87 kilodaltons (87 kDa) was previously identified as a major specific substrate for protein kinase C in neuronal and other tissues. We have now studied the effect of protein kinase C-catalyzed phosphorylation of this protein on its association with membranes in isolated nerve terminals (synaptosomes) from rat cerebral cortex. Incubation of synaptosomal membranes under conditions associated with activation of protein kinase C led to the release of the phosphorylated 87-kDa protein into the incubation medium. In intact synaptosomes, activation of protein kinase C by phorbol esters or by depolarization-induced Ca2+ influx caused an increased phosphorylation of the 87-kDa protein and its translocation from membrane to cytosol. This translocation showed time courses, calcium dependency, and reversibility similar to those observed for the protein kinase C-induced phosphorylation of the protein. These results suggest that protein kinase C-catalyzed phosphorylation of the 87-kDa protein is responsible for its subcellular translocation into the cytosol of nerve terminals.

TPA induces subcellular translocation and subsequent down-regulation of both phorbol ester binding and protein kinase C activities in MCF-7 cells.

Phorbol ester TPA has been previously shown to induce a rapid translocation, followed by a progressive decline of protein kinase C activity in MCF-7 cells (J.M. Darbon et al, 1986, Biochem. Biophys. Res. Comm. 137: 1159-1166). We show now a parallel TPA-induced movement of phorbol ester binding sites from the cytosolic to the particulate fraction with no change in the binding affinities for the (3H) PDBu probe (KD congruent to 2 nM). The subcellular redistribution process is followed by a rapid decrease of the phorbol ester binding capacity at the membrane level. The concomitant decline in both phorbol ester binding and protein kinase C activities that we observed during the course of TPA treatment strongly argues for a real down-regulation of the enzyme in phorbol ester-treated MCF-7 cells. The molecular mechanisms of these events and their relations to the inhibition of cell growth remain to be clarified.

Phosphorylation/dephosphorylation of the beta-adrenergic receptor regulates its functional coupling to adenylate cyclase and subcellular distribution.

Prolonged exposure of cells or tissues to drugs or hormones such as catecholamines leads to a state of refractoriness to further stimulation by that agent, known as homologous desensitization. In the case of the beta-adrenergic receptor coupled to adenylate cyclase, this process has been shown to be intimately associated with the sequestration of the receptors from the cell surface through a cAMP-independent process. Recently, we have shown that homologous desensitization in the frog erythrocyte model system is also associated with increased phosphorylation of the beta-adrenergic receptor. We now provide evidence that the phosphorylation state of the beta-adrenergic receptor regulates its functional coupling to adenylate cyclase, subcellular translocation, and recycling to the cell surface during the process of agonist-induced homologous desensitization. Moreover, we show that the receptor phosphorylation is reversed by a phosphatase specifically associated with the sequestered subcellular compartment. At 23 degrees C, the time courses of beta-adrenergic receptor phosphorylation, sequestration, and adenylate cyclase desensitization are identical, occurring without a lag, exhibiting a t1/2 of 30 min, and reaching a maximum at approximately 3 hr. Upon cell lysis, the sequestered beta-adrenergic receptors can be partially recovered in a light membrane vesicle fraction that is separable from the plasma membranes by differential centrifugation. The increased beta-adrenergic receptor phosphorylation is apparently reversed in the sequestered vesicle fraction as the sequestered receptors exhibit a phosphate/receptor stoichiometry that is similar to that observed under basal conditions. High levels of a beta-adrenergic receptor phosphatase activity appear to be associated with the sequestered vesicle membranes. The functional activity of the phosphorylated beta-adrenergic receptor was examined by reconstituting purified receptor with its biochemical effector the guanine nucleotide regulatory protein (Ns) in phospholipid vesicles and assessing the receptor-stimulated GTPase activity of Ns. Compared to controls, phosphorylated beta-adrenergic receptors, purified from desensitized cells, were less efficacious in activating the Ns GTPase activity. These results suggest that phosphorylation of the beta-adrenergic receptor leads to its functional uncoupling and physical translocation away from the cell surface into a sequestered membrane domain. In the sequestered compartment, the phosphorylation is reversed thus enabling the receptor to recycle back to the cell surface and recouple with adenylate cyclase.

Translocation of protein kinase C in human leukemia cells susceptible or resistant to differentiation induced by phorbol 12-myristate 13-acetate.

We investigated the possible relationship between the susceptibility of cells to differentiation induced by phorbol 12-myristate 13-acetate (PMA) and the subcellular translocation of calcium- and phospholipid-dependent protein kinase (protein kinase C) activity from the cytosol to the membrane. These two events were analyzed in a number of human leukemia cell lines, including four cell variants of the promyelocytic cell line HL-60 that exhibit different degrees of susceptibility to PMA-induced differentiation. The phenotype of the differentiated cells was characterized by increased reactivity with monoclonal antibodies against maturation-specific cell surface antigens, increased nonspecific esterase activity, and acquisition of morphological cell maturation. Analysis of the subcellular distribution of protein kinase C activity in each of these cell types revealed that 90% of the kinase activity was present in the cytosolic fraction, with the remaining activity in the membrane fraction. Treatment of the differentiation-susceptible cells with 160 nM PMA resulted, within 5 min after treatment, in a greater than 60% decrease in protein kinase C activity in the cytosolic fraction and a greater than 1500% increase in the activity in the membrane fraction. No such subcellular redistribution of protein kinase C activity was found after treatment of the differentiation-resistant cells. On the basis of these findings, we suggest that the process of subcellular translocation of protein kinase C activity, initiated after the binding of PMA to this kinase, is required for the induction of cell differentiation by this phorbol diester.

Activation by phorbol esters of protein kinase C in MCF-7 human breast cancer cells

Exposure of MCF-7 human breast cancer cells to the phorbol ester 12- O-tetradecanoylphorbol-13-acetate (TPA) leads to the inhibition of cell proliferation. We investigate here the short-term effects of TPA on subcellular distribution of protein kinase C, and on protein phosphorylation in cultured MCF-7 cells. We report a rapid and dramatic decrease in cytosolic protein kinase C activity after TPA treatment. Only 30% of the enzymatic activity lost in the cytosol was recovered in the particulate fraction. These data suggest that subcellular translocation of protein kinase C is accompanied by a rapid down-regulation of the enzyme (70%). Furthermore, TPA and other protein kinase C activators rapidly induce the phosphorylation of a 28 kDa protein in intact MCF-7 cells. Phorbol esters devoid of tumor-promoting activity are ineffective both for inducing these early biochemical events and for inhibiting cell proliferation.

Subcellular translocation of glucose transporters: role in insulin action and its perturbation in altered metabolic states.

In this article we have described the hypothesis that insulin stimulates glucose transport through glucose transporter translocation from an intracellular pool to the plasma membrane. In addition, we have shown that changes in the numbers and subcellular distributions of glucose transporters correlate with alterations in insulin-stimulated glucose transport activity in several experimental models of insulin resistance and hyperresponsiveness. However, in experiments with counterregulatory hormones and with hyperresponsive states induced by nutritional repletion following deprivation, changes in insulin responsiveness cannot be fully explained by such alterations in the numbers and/or subcellular distribution of glucose transporters. Thus, evidence has been presented for changes in glucose transporter intrinsic activity that both inhibit and augment insulin-stimulated glucose transport rates. Finally, we have discussed data suggesting that the translocation process is applicable to human tissue and that significant changes in adipose cell glucose transport activity have been correlated with total glucose disposal in various metabolic states in humans. Determining the physiologic factors involved in modulating these events at the cellular level is an important area for further investigation.

Insulin-induced internalization of the insulin receptor in the isolated rat adipose cell. Detection of both major receptor subunits following their biosynthetic labeling in culture.

A protocol has been developed for maintaining isolated rat adipose cells in primary tissue culture. Using this protocol, cells remain fully viable and responsive to insulin for at least 24 h, as assessed by measuring 3-0-methylglucose transport, lipogenesis from [U-14C]glucose, and the incorporation of [35S]methionine into total membrane protein. The acute insulin-induced internalization of its own receptor was then examined by biosynthetically labeling cells in culture with either [35S]methionine or [3H]glucosamine, maximally inducing receptor internalization with a 30-min incubation in the presence of saturating insulin, and preparing plasma and low-density microsomal membrane fractions by differential ultracentrifugation. Receptors were immunoprecipitated with anti-receptor antiserum, and the receptor subunits separated by NaDodSO4-PAGE under reducing conditions and analyzed by autoradiography. When cells not acutely treated with insulin are examined, both the 135K alpha- and 95K beta-receptor subunits are prominently labeled in the plasma membrane fraction, but only faintly labeled in the low-density microsomal membrane fraction. Following the induction of maximal acute receptor internalization, both subunits are decreased by 20-30% in the plasma membrane fraction and concomitantly increased in the low-density microsomal membrane fraction. However, the relative molecular weights and labeling intensities of the two subunits remain constant and correspond to those observed in the biosynthetically labeled human lymphocyte receptor. A minor band of Mr congruent to 190K is also labeled, but its labeling intensity is similar in the two membrane fractions from basal cells and does not change in response to insulin.(ABSTRACT TRUNCATED AT 250 WORDS)