Rapamycin-sensitive phosphorylation of PKC on a carboxy-terminal site by an atypical PKC complex

Ziegler, Wolfgang; Parekh, Davey B.; Good, J. Ann Le; Whelan, Richard D. H.; Kelly, J.J.; Frech, Matthias; Hemmings, Brian A.; Parker, Peter J.

doi:10.1016/s0960-9822(99)80236-x

Cited by 100 publications

(99 citation statements)

References 33 publications

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“…Amino acids have a similar, although less potent, effect on insulin release. Moreover, an amino acid-sensing pathway also promotes the formation of competent enzyme by activating mTOR, which is thought to prevent dephosphorylation of the hydrophobic site Ser-660, which is crucial for PKC ␤II activity, by inhibiting a phosphatase (12,18,19,29). Thus, perfusion of jejunum with either wortmannin or rapamycin in the presence of fructose blocks the formation of competent PKC ␤II and the whole of the signaling pathway (Figs.…”

Section: Discussionmentioning

confidence: 99%

“…Activation of PKC ␤II is tightly coupled to phosphorylation of the hydrophobic site Ser-660, which greatly increases affinity for Ca 2ϩ (17). Phosphorylation of the hydrophobic site in PKC ␦ and PKC ⑀ is sensitive to rapamycin (18,19), suggesting a role for mTOR in controlling phosphorylation at this site, possibly by regulating the activity of a phosphatase (12).…”

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confidence: 99%

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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: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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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“…PKCe has been shown to be phosphorylated by PDK-1 in vitro and in cells (Le Good et al, 1998). PDK-1 phosphorylates PKCe at the activation-loop residue Thr566 and Ser729, leading to its activation Ziegler et al, 1999;Cenni et al, 2002). Although PDK-1 has been reported to be constitutively activated in cells, it is hypothesized that upon stimulation of cells by growth factors, PDK-1 is recruited to the membrane due to binding of phosphatidylinositol-3,4,5-triphosphate to its PH domain (Anderson et al, 1998).…”

Section: Discussionmentioning

confidence: 99%

Expression of p21WAF1/Cip1 through Sp1 sites by histone deacetylase inhibitor apicidin requires PI 3-kinase–PKCε signaling pathway

et al. 2003

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We previously reported that the activation of p21 WAF1/Cip1 transcription by histone deacetylase inhibitor apicidin was mediated through Sp1 sites and pointed to the possible participation of protein kinase C (PKC). In this study, we investigated the role and identity of the specific isoforms of PKC involved and identified phosphatidylinositol 3-kinase (PI 3-kinase) as an upstream effector in HeLa cells. Using an isoform-specific pharmacological inhibitor of PKC, a PKCe dominant-negative mutant, and antisense oligonucleotide to inhibit PKCe specifically, we found that among PKC isoforms, PKCe was required for the p21 WAF1/ Cip1 expression by apicidin. In addition to PKCe, PI 3-kinase appeared to participate in the activation of p21 WAF1/Cip1 promoter by apicidin, since inactivation of PI 3-kinase either by transient expression of dominantnegative mutant of PI 3-kinase or its specific inhibitors, LY294002 and wortmannin, attenuated the activation of p21 WAF1/Cip1 promoter and p21 WAF1/Cip1 protein expression by apicidin. Furthermore, membrane translocation of PKCe in response to apicidin was blocked by the PI 3-kinase inhibitor, indicating the role of PI 3-kinase as an upstream molecule of PKCe in the p21 WAF1/Cip1 promoter activation by apicidin. However, the p21 WAF1/Cip1 expression by apicidin appeared to be independent of the histone hyperacetylation, since apicidin-induced histone hyperacetylation of p21 WAF1/Cip1 promoter region was not affected by inhibition of PI 3-kinase and PKC, suggesting that the chromatin remodeling through the histone hyperacetylation alone might not be sufficient for the expression of p21 WAF1/Cip1 by apicidin. Taken together, these results suggest that the PI 3-kinase-PKCe signaling pathway plays a pivotal role in the expression of the p21 WAF1/Cip1 by apicidin.

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“…Assuming both activities are physiologically meaningful, such a breadth in substrate specificity is relatively unprecedented among the protein kinases. Finally, it should be noted that Phe-Ser/Thr-Phe/Tyr motifs homologous to p70␣ Thr-412 are found in many kinases of the AGC (protein kinase A, G, and C) subclass, and it is of interest to note the recent report that PKC␦ activation and phosphorylation at Ser-662 (in the context Phe-Ser-Phe) is inhibitable by rapamycin (23). In conclusion, the present study demonstrates the in vitro activation of p70␣ by mTOR-catalyzed phosphorylation involving p70␣ Thr-412, a critical site conserved in the other AGC kinase subfamily members.…”

Section: Fig 2 Phosphorylation Of P70␣ Mutants By Mtormentioning

confidence: 99%

Immunopurified Mammalian Target of Rapamycin Phosphorylates and Activates p70 S6 Kinase α in Vitro

Isotani

Hara

Tokunaga

et al. 1999

Journal of Biological Chemistry

312

246

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p70 S6 kinase ␣ (p70␣) is activated in vivo through a multisite phosphorylation in response to mitogens if a sufficient supply of amino acids is available or to high concentrations of amino acids per se. The immunosuppressant drug rapamycin inhibits p70␣ activation in a manner that can be overcome by coexpression of p70␣ with a rapamycin-resistant mutant of the mammalian target of rapamycin (mTOR) but only if the mTOR kinase domain is intact. We report here that a mammalian recombinant p70␣ polypeptide, extracted in an inactive form from rapamycin-treated cells, can be directly phosphorylated by the mTOR kinase in vitro predominantly at the rapamycin-sensitive site Thr-412. mTOR-catalyzed p70␣ phosphorylation in vitro is accompanied by a substantial restoration in p70␣ kinase activity toward its physiologic substrate, the 40 S ribosomal protein S6. Moreover, sequential phosphorylation of p70␣ by mTOR and 3-phosphoinositide-dependent protein kinase 1 in vitro resulted in a synergistic stimulation of p70␣ activity to levels similar to that attained by serum stimulation in vivo. These results indicate that mTOR is likely to function as a direct activator of p70 in vivo, although the relative contribution of mTOR-catalyzed p70 phosphorylation in each of the many circumstances that engender p70 activation remains to be defined. p70 S6 kinase ␣ (p70␣), 1 whose major substrate is the 40 S ribosomal protein S6, plays a critical role in the translation of a subclass of mRNAs that contain a short oligopyrimidine sequence immediately following the transcriptional start site (1). p70␣ is activated in response to insulin/mitogens in vivo through a multisite phosphorylation of serine and threonine residues (2). Several sets of independently regulated p70␣ phosphorylation sites have been identified (3-6); one set consists of Ser/Thr-Pro motifs, five of which are clustered in a psuedosubstrate autoinhibitory domain in the noncatalytic carboxyl-terminal tail (Ser-434, Ser-441, Ser-447, Ser-452, and Thr-444 in p70␣), and two others, Thr-390 and Ser-394, are located in a 65-amino acid segment immediately carboxyl-terminal to the kinase catalytic domain. A second set of regulated phosphorylation sites, Thr-412 and Ser-427, exhibit the sequence motif Phe-Ser/Thr-Phe/Tyr. Thr-252, located on the activation loop in catalytic subdomain VIII, is the site at which 3-phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates p70␣ (7,8). Among these, the phosphorylation of Thr-252, Ser-394, and Thr-412 is necessary for the activation of p70␣ kinase catalytic function; the attainment of physiologic levels of p70␣ activity results from a strongly synergistic, positive site-site interaction between the phosphorylated Thr-252 and Thr-412 residues (7).In addition to its regulation by insulin and mitogens through PI-3 kinase-dependent pathways, p70␣ can also be activated by increasing concentrations of extracellular amino acids in the absence of serum or mitogens to the level attained by maximal mitogen stimulation (9-11). Moreover, a thr...

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Rapamycin-sensitive phosphorylation of PKC on a carboxy-terminal site by an atypical PKC complex

Abstract: PKCzeta is identified as a component of the upstream kinase responsible for the phosphorylation of the PKCdelta hydrophobic site in vitro and in vivo. PKCzeta can therefore control the phosphorylation of this PKCdelta site, antagonizing a rapamycin-sensitive pathway.

Cited by 100 publications

References 33 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

Expression of p21WAF1/Cip1 through Sp1 sites by histone deacetylase inhibitor apicidin requires PI 3-kinase–PKCε signaling pathway

Immunopurified Mammalian Target of Rapamycin Phosphorylates and Activates p70 S6 Kinase α in Vitro

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