Multisite dephosphorylation and desensitization of conventional protein kinase C isotypes

Hansra, Gurdip; Garcia-Paramio, Pilar; Prévostel, Corinne; Whelan, Richard D. H.; Bornancin, Frédéric; Parker, Peter J.

doi:10.1042/bj3420337

Cited by 116 publications

(79 citation statements)

References 47 publications

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“…Since the 42-kDa species is derived by dephosphorylation of the 49-kDa species (Fig. 3), it seems that proteasomal degradation follows dephosphorylation in agreement with Hansra et al (24). The order of production of detected species from the 80-/78-kDa native enzyme therefore appears to be 49, 42, 180, and 250ϩ kDa.…”

Section: Resultssupporting

confidence: 72%

“…3). That activation at the membrane results ultimately in dephosphorylation is in keeping with the fact that activation induces an open conformation, which increases the sensitivity of the C terminus to dephosphorylation by more than 2 orders of magnitude (24). In addition, the isolated catalytic domain naturally has an open conformation (14,25).…”

Section: Resultsmentioning

confidence: 70%

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Intestinal Sugar Absorption Is Regulated by Phosphorylation and Turnover of Protein Kinase C βII Mediated by Phosphatidylinositol 3-Kinase- and Mammalian Target of Rapamycin-dependent Pathways

Helliwell

Rumsby

Kellett

2003

Journal of Biological Chemistry

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Stimulation of intestinal fructose absorption by phorbol 12-myristate 13-acetate (PMA) results from rapid insertion of GLUT2 into the brush-border membrane and correlates with protein kinase C (PKC) ␤II activation. We have therefore investigated the role of phosphatidylinositol 3 (PI3)-kinase and mammalian target of rapamycin in the regulation of fructose absorption by PKC ␤II phosphorylation. In isolated jejunal loops, stimulation of fructose absorption by PMA was inhibited by preperfusion with wortmannin or rapamycin, which blocked GLUT2 activation and insertion into the brushborder membrane. Antibodies to the last 18 and last 10 residues of the C-terminal region of PKC ␤II recognized several species differentially in Western blots. Extensive cleavage of native enzyme (80/78 kDa) to a catalytic domain product of 49 kDa occurred. PMA and sugars provoked turnover and degradation of PKC ␤II by dephosphorylation to a 42-kDa species, which was converted to polyubiquitylated species detected at 180 and 250؉ kDa. PMA increased the level of the PKC ␤II 49-kDa species, which correlates with the GLUT2 level; wortmannin and rapamycin blocked these effects of PMA. Rapamycin and wortmannin inhibited PKC ␤II turnover. PI3-kinase, PDK-1, and protein kinase B were present in the brush-border membrane, where their levels were increased by PMA and blocked by the inhibitors. We conclude that GLUT2-mediated fructose absorption is regulated through PI3-kinase and mammalian target of rapamycin-dependent pathways, which control phosphorylation of PKC ␤II and its substrate-induced turnover and ubiquitin-dependent degradation. These findings suggest possible mechanisms for short term control of intestinal sugar absorption by insulin and amino acids.Intestinal glucose absorption comprises two components (for a review, see Ref. 1). It is well established that one of these is an active component mediated by the Na ϩ /glucose cotransporter, SGLT1. We have proposed that the other is a facilitated component mediated by glucose-dependent activation and recruitment of GLUT2 to the brush-border membrane. GLUT2 recruitment correlates with SGLT1-dependent activation of PKC 1 ␤II (2) so that SGLT1 is now seen to exert an important regulatory role in addition to its established functions as a scavenger and transporter (1). Regulation of the facilitated component may also involve mitogen-activated protein (MAP) kinase-and PI3-kinase-dependent signaling pathways (3).The K a and J max of the facilitated component are 2-and 3-fold, respectively, of those for SGLT1 in vivo. After a meal, the average concentration across the luminal contents of rat jejunum reaches 48 mM (4), close to the K m of the facilitated component, and much higher concentrations may well be present locally at the brush-border membrane, as a result of the hydrolysis of complex dietary sugars (5). The facilitated component therefore appears to provide the major route by which glucose is absorbed immediately after a meal. Moreover, the activation of GLUT2 and its rapid trafficking to...

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Section: Resultssupporting

confidence: 72%

Section: Resultsmentioning

confidence: 70%

Intestinal Sugar Absorption Is Regulated by Phosphorylation and Turnover of Protein Kinase C βII Mediated by Phosphatidylinositol 3-Kinase- and Mammalian Target of Rapamycin-dependent Pathways

Helliwell

Rumsby

Kellett

2003

Journal of Biological Chemistry

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“…The phosphorylations act cooperatively to maintain the active (latent) conformation. The phosphorylated species is found in the cytosol or, on activation, is associated with membranes (26). PKC signaling is desensitized through dephosphorylation and proteolysis.…”

Section: Discussionmentioning

confidence: 99%

Distinct kinases are involved in contraction of cat esophageal and lower esophageal sphincter smooth muscles

Kim

Cao

Song

et al. 2004

American Journal of Physiology-Cell Physiology

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Biancani. Distinct kinases are involved in contraction of cat esophageal and lower esophageal sphincter smooth muscles. Am J Physiol Cell Physiol 287: C384 -C394, 2004; 10.1152/ajpcell.00390.2003.-Contraction of smooth muscle depends on the balance of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. Because MLCK activation depends on the activation of calmodulin, which requires a high Ca 2ϩ concentration, phosphatase inhibition has been invoked to explain contraction at low cytosolic Ca 2ϩ levels. The link between activation of the Ca 2ϩ -independent protein kinase C⑀ (PKC⑀) and MLC phosphorylation observed in the esophagus (ESO) (Sohn UD, Cao W, Tang DC, Stull JT, Haeberle JR, Wang CLA, Harnett KM, Behar J, and Biancani P. Am J Physiol Gastrointest Liver Physiol 281: G467-G478, 2001), however, has not been elucidated. We used phosphatase and kinase inhibitors and antibodies to signaling enzymes in combination with intact and saponin-permeabilized isolated smooth muscle cells from ESO and lower esophageal sphincter (LES) to examine PKC⑀-dependent, Ca 2ϩ -independent signaling in ESO. The phosphatase inhibitors okadaic acid and microcystin-LR, as well as an antibody to the catalytic subunit of type 1 protein serine/threonine phosphatase, elicited similar contractions in ESO and LES. MLCK inhibitors (ML-7, ML-9, and SM-1) and antibodies to MLCK inhibited contraction induced by phosphatase inhibition in LES but not in ESO. The PKC inhibitor chelerythrine and antibodies to PKC⑀, but not antibodies to PKC␤II, inhibited contraction of ESO but not of LES. In ESO, okadaic acid triggered translocation of PKC⑀ from cytosolic to particulate fraction and increased activity of integrin-linked kinase (ILK). Antibodies to the mitogen-activated protein (MAP) kinases ERK1/ERK2 and to ILK, and the MAP kinase kinase (MEK) inhibitor PD-98059, inhibited okadaic acid-induced ILK activity and contraction of ESO. We conclude that phosphatase inhibition potentiates the effects of MLCK in LES but not in ESO. Contraction of ESO is mediated by activation of PKC⑀, MEK, ERK1/2, and ILK. protein kinase C; myosin light chain kinase; phosphatase; integrinlinked kinase SMOOTH MUSCLE CONTRACTION is regulated primarily by the reversible phosphorylation of myosin (1, 27, 58), depending on the balance between myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities, and both enzymes are subject to regulation. Most contractile stimuli elicit an increase in intracellular free Ca 2ϩ concentration ([Ca 2ϩ ] i ) via entry of Ca 2ϩ from the extracellular space and/or release from intracellular stores, whereupon Ca 2ϩ activates calmodulin (CaM)-dependent MLCK. Activated MLCK phosphorylates the 20-kDa myosin light chains (MLC) of myosin II at Ser 19 to activate cross-bridge cycling and the development of force or shortening of the muscle. Relaxation generally follows removal of the stimulus, whereupon [Ca 2ϩ ] i returns to resting levels as Ca 2ϩ is removed from the cytosol. MLCK (32) is i...

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“…These phosphorylation sites are conserved among all isoforms except and (32). Phosphorylation of PKC is essential for optimal catalytic activity (31,34), and dephosphorylation has been suggested to prime PKC for degradation (35). There is also evidence that autophosphorylation influences PKC localization (36).…”

Section: Discussionmentioning

confidence: 99%

Regulation of Protein Kinase C by the Cytoskeletal Protein Calponin

Leinweber

Parissenti

Gallant

et al. 2000

Journal of Biological Chemistry

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Previous studies from this laboratory have shown that, upon agonist activation, calponin co-immunoprecipitates and co-localizes with protein kinase C⑀ (PKC⑀) in vascular smooth muscle cells. In the present study we demonstrate that calponin binds directly to the regulatory domain of PKC both in overlay assays and, under native conditions, by sedimentation with lipid vesicles. Calponin was found to bind to the C2 region of both PKC⑀ and PKC␣ with possible involvement of C1B. The C2 region of PKC⑀ binds to the calponin repeats with a requirement for the region between amino acids 160 and 182. We have also found that calponin can directly activate PKC autophosphorylation. By using anti-phosphoantibodies to residue Ser-660 of PKC␤II, we found that calponin, in a lipid-independent manner, increased auto-phosphorylation of PKC␣, -⑀, and -␤II severalfold compared with control conditions. Similarly, calponin was found to increase the amount of 32 P-labeled phosphate incorporated into PKC from [␥-32 P]ATP. We also observed that calponin addition strongly increased the incorporation of radiolabeled phosphate into an exogenous PKC peptide substrate, suggesting an activation of enzyme activity. Thus, these results raise the possibility that calponin may function in smooth muscle to regulate PKC activity by facilitating the phosphorylation of PKC.

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Multisite dephosphorylation and desensitization of conventional protein kinase C isotypes

Cited by 116 publications

References 47 publications

Intestinal Sugar Absorption Is Regulated by Phosphorylation and Turnover of Protein Kinase C βII Mediated by Phosphatidylinositol 3-Kinase- and Mammalian Target of Rapamycin-dependent Pathways

Intestinal Sugar Absorption Is Regulated by Phosphorylation and Turnover of Protein Kinase C βII Mediated by Phosphatidylinositol 3-Kinase- and Mammalian Target of Rapamycin-dependent Pathways

Distinct kinases are involved in contraction of cat esophageal and lower esophageal sphincter smooth muscles

Regulation of Protein Kinase C by the Cytoskeletal Protein Calponin

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