Antipeptide antibodies directed against the C‐terminus of protein kinase Cζ (PKCζ) react with a Ca<sup>2+</sup>‐ and TPA‐sensitive PKC in HT‐29 human intestinal epithelial cells

Batlle, Eduard; Fabre, Myriam; Herreros, Antonio Garcı́a de

doi:10.1016/0014-5793(94)00379-3

Cited by 16 publications

(13 citation statements)

References 24 publications

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“…The highest molecular weight band (C, 80 kD) seen on this blot may represent a conventional isozyme, possibly PKC-ex; cross-reactivity of C-terminal C antibodies with conventional isozymes has been reported previously. (30,31) As was observed with the PKC-exspecific antibody (Fig. lA), this isozyme was expressed in each of the osteoblasts screened.…”

Section: Pkc Isozyme Expression In Normal Osteoblasts and Osteoblastisupporting

confidence: 62%

Expression and phorbol ester–induced down-regulation of protein kinase C isozymes in osteoblasts

Sanders

Stern

1996

Journal of Bone and Mineral Research

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The protein kinase C (PKC) enzyme family consists of at least 11 isozymes in three classes, with characteristic tissue distributions. Phorbol esters activate and ultimately down-regulate phorbol-sensitive isozymes. PKC is a signal transducer in bone, and phorbol esters influence bone resorption. Little is known about specific PKC isozymes in this tissue, however. We describe here the expression and phorbol ester-induced down-regulation of PKC isozymes in osteoblasts. Normal mouse osteoblasts and seven osteoblastic cell lines (rat UMR-106, ROS 17/2.8, ROS 24/1, and human MG-63, G-292, SaOS-2, HOS-TE85) were screened for isozyme expression by Western immunoblotting using isozyme-specific anti-PKC antibodies. The conventional alpha and beta I isozymes, but not gamma, were present in each of the osteoblasts examined; PKC-beta II was detectable in all but the ROS 24/1 line. PKC-epsilon was expressed in all osteoblasts screened, but other novel PKCs, delta, eta, and theta, were detectable only in select lines. The atypical zeta and iota/lambda PKCs were in all osteoblasts examined. To determine the sensitivity of the isozymes to prolonged phorbol ester treatment, normal osteoblasts and the UMR-106 cell line were treated with vehicle or 1 microM phorbol 12, 13-dibutyrate (PDB) for 1, 3, 6, 12, 24, or 48 h, and Western blot analysis was performed. Normal and UMR-106 cells showed similar phorbol sensitivities; conventional (alpha, beta I) and novel (delta, epsilon, eta) isozymes were down-regulated by prolonged phorbol treatment but atypical isozymes were not. Down-regulation of all sensitive PKCs was detectable within 6 h of phorbol treatment; the novel delta and epsilon isozymes, however, showed more rapid and dramatic down-regulation than conventional isozymes. The observed down-regulation was dose-dependent (0.3-3 microM) and specific; 48 h treatment with the inactive phorbol, 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), failed to down-regulate PDB-sensitive isozymes. The phorbol-induced down-regulation was also reversible; 24 h after withdrawing PDB, all phorbol-sensitive isozymes, except PKC-eta, had recovered at least partially. These studies, the first to characterize thoroughly PKC isozyme expression in osteoblastic cells from several species, demonstrate that osteoblasts have a characteristic PKC isozyme profile, including both phorbol ester-sensitive and -insensitive isozymes. The time course of down-regulation and the presence of phorbol-insensitive PKCs must be considered in interpreting the effects of phorbol esters on bone remodeling.

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Section: Pkc Isozyme Expression In Normal Osteoblasts and Osteoblastisupporting

confidence: 62%

Expression and phorbol ester–induced down-regulation of protein kinase C isozymes in osteoblasts

Sanders

Stern

1996

Journal of Bone and Mineral Research

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“…The third antibody (Upstate Biotechnology) used in the present study is reported not to cross-react with either PKC-␣, PKC-, or PKC-(according to information provided by the manufacturer). It has also been reported that antibodies directed against the COOH terminus of PKC-react with a Ca 2ϩ and phorbol ester-sensitive PKC isoform (25). However, the kinase that is the subject of this report is only weakly activated by a combination of PMA, Ca 2ϩ , and PS and has sustained activity in the absence of exogenous lipids.…”

Section: Fig 6 Abrogation Of Lps-induced Activation Of Pkc-by the Pmentioning

confidence: 44%

Phosphatidylinositol 3-Kinase-dependent Activation of Protein Kinase C-ζ in Bacterial Lipopolysaccharide-treated Human Monocytes

Herrera-Velit¹,

Knutson²,

Reiner³

1997

Journal of Biological Chemistry

131

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The isoform identity of activated protein kinase C (PKC) and its regulation were investigated in bacterial lipopolysaccharide (LPS)-treated human monocytes. Resolution of detergent-soluble lysates prepared from LPS-treated, peripheral blood monocytes using Mono Q anion-exchange chromatography revealed two principal peaks of myelin basic protein kinase activity. Immunoblotting and immunoprecipitation with isoform-specific anti-PKC antibodies showed that the major and latest eluting peak is accounted for by PKC-. In addition to primary monocytes, activation of PKCin response to LPS was also observed in the human promonocytic cell lines, U937 and THP-1. Consistent with its identity as PKC-, the kinase did not depend upon the presence of lipids, Ca 2؉ , or diacylglycerol for activity. In addition, the kinase phosphorylates peptide ⑀ and myelin basic protein with equal efficiency but phosphorylates Kemptide and protamine sulfate poorly. Translocation of PKC-from the cytosolic to the particulate membrane fraction upon exposure of monocytes to LPS provided further evidence for activation of the kinase.Preincubation of monocytes with the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors, wortmannin or LY294002, abrogated LPS-induced activation of PKC-. Furthermore, activation of PKC-failed to occur in U937 cells transfected with a dominant negative mutant of the p85 subunit of PI 3-kinase. PKC-activity was also observed to be enhanced in vitro by the addition of phosphatidylinositol 3,4,5P 3 . These findings are consistent with a model in which PKC-is activated downstream of PI 3-kinase in monocytes in response to LPS.

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“…in subsequent immunoblots using PKC £ and PKC e antibodies, since the other apparently specific signals of 82 kDa and 80 kDa represent conventional PKC protein (29,30) and PKC 8 (C.S.-P, C.L.B., T.J.B., unpublished observations), respectively. Other PKC isozymes were not quantified: PKC P immunoreactivity was extremely low (not shown), using any of three PKC 3-specific antibodies, making densitometry impracticable, while PKC 7, PKC r\, and PKC 1 are absent or only poorly expressed in skeletal muscle (24,31,32).…”

Section: Resultsmentioning

confidence: 97%

Alterations in the Expression and Cellular Localization of Protein Kinase C Isozymes ε and θ Are Associated With Insulin Resistance in Skeletal Muscle of the High-Fat–Fed Rat

et al. 1997

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We have tested the hypothesis that changes in the levels and cellular location of protein kinase C (PKC) isozymes might be associated with the development of insulin resistance in skeletal muscles from the high-fat-fed rat. Lipid measurements showed that triglyceride and diacylglycerol, an activator of PKC, were elevated four- and twofold, respectively. PKC activity assays indicated that the proportion of membrane-associated calcium-independent PKC was also increased. As determined by immunoblotting, total (particulate plus cytosolic) PKC alpha, epsilon, and zeta levels were not different between control and fat-fed rats. However, the ratio of particulate to cytosolic PKC epsilon in red muscles from fat-fed rats was increased nearly sixfold, suggesting chronic activation. In contrast, the amount of cytosolic PKC theta was downregulated to 45% of control, while the ratio of particulate to cytosolic levels increased, suggesting a combination of chronic activation and downregulation. Interestingly, while insulin infusion in glucose-clamped rats increased the proportion of PKC theta in the particulate fraction of red muscle, this was potentiated by fat-feeding, suggesting that the translocation is a consequence of altered lipid flux rather than a proximal event in insulin signaling. PKC epsilon and theta measurements from individual rats correlated with triglyceride content of red gastrocnemius muscle; they did not correlate with plasma glucose, which was not elevated in fat-fed rats, suggesting that they were not simply a consequence of hyperglycemia. Our results suggest that these specific alterations in PKC epsilon and PKC theta might contribute to the link between increased lipid availability and muscle insulin resistance previously described using high-fat-fed rats.

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Antipeptide antibodies directed against the C‐terminus of protein kinase Cζ (PKCζ) react with a Ca²⁺‐ and TPA‐sensitive PKC in HT‐29 human intestinal epithelial cells

Cited by 16 publications

References 24 publications

Expression and phorbol ester–induced down-regulation of protein kinase C isozymes in osteoblasts

Expression and phorbol ester–induced down-regulation of protein kinase C isozymes in osteoblasts

Phosphatidylinositol 3-Kinase-dependent Activation of Protein Kinase C-ζ in Bacterial Lipopolysaccharide-treated Human Monocytes

Alterations in the Expression and Cellular Localization of Protein Kinase C Isozymes ε and θ Are Associated With Insulin Resistance in Skeletal Muscle of the High-Fat–Fed Rat

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Antipeptide antibodies directed against the C‐terminus of protein kinase Cζ (PKCζ) react with a Ca2+‐ and TPA‐sensitive PKC in HT‐29 human intestinal epithelial cells

Cited by 16 publications

References 24 publications

Expression and phorbol ester–induced down-regulation of protein kinase C isozymes in osteoblasts

Expression and phorbol ester–induced down-regulation of protein kinase C isozymes in osteoblasts

Phosphatidylinositol 3-Kinase-dependent Activation of Protein Kinase C-ζ in Bacterial Lipopolysaccharide-treated Human Monocytes

Alterations in the Expression and Cellular Localization of Protein Kinase C Isozymes ε and θ Are Associated With Insulin Resistance in Skeletal Muscle of the High-Fat–Fed Rat

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Antipeptide antibodies directed against the C‐terminus of protein kinase Cζ (PKCζ) react with a Ca²⁺‐ and TPA‐sensitive PKC in HT‐29 human intestinal epithelial cells