A synthetic peptide substrate for selective assay of protein kinase C

Yasuda, Ichiro; Kishimoto, Akira; Tanaka, Shinichiro; Tominaga, Masahiro; Sakurai, Atsushi; Nishizuka, Yasutomi

doi:10.1016/0006-291x(90)90996-z

Cited by 293 publications

(154 citation statements)

References 26 publications

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“…PMA (phorbol 12-myristate 13-acetate)-activated PKC activity was assayed by phosphorylation of an acetylated peptide based on myelin basic protein, Ac-MBP (4-14), (Yashuda et al, 1990), as described previously (Prendiville et al, 1994). P388 cells (approximately 1 x 107 per assay) were rapidly pelleted, washed with icecold phosphate-buffered saline, resuspended in ice-cold extraction buffer (20 mm Tris, pH 7.5, 0.5 mM EDTA, 0.5% Triton X-100 and (Steel and Peckham, 1979) 25 ug ml-1 aprotinin and leupeptin) and sonicated.…”

Section: Methodsmentioning

confidence: 99%

Bryostatin 1-tamoxifen combinations show synergistic effects on the inhibition of growth of P388 cells in vitro

McGown

Jayson²,

Pettit³

et al. 1998

Br J Cancer

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Summary This study shows that combinations of bryostatin 1, a novel modulator of protein kinase C currently under clinical evaluation, with the anti-oestrogenic agent tamoxifen caused a large synergistic enhancement of growth inhibition in P388 cells in vitro. The growth-inhibitory effects of bryostatin 1 in the presence of non-inhibitory concentrations of tamoxifen were increased by approximately 200-fold, whereas growth inhibition by tamoxifen in the presence of non-inhibitory concentrations of bryostatin 1 were increased over 30-fold. These data have been confirmed by isobologram analysis. The precise mechanism underlying this effect is unknown, although preliminary data implicating protein kinase C is presented. The magnitude of this synergistic effect, together with evidence of clinical responses seen when these agents were given sequentially in ovarian cancer, merits further study.

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Section: Methodsmentioning

confidence: 99%

Bryostatin 1-tamoxifen combinations show synergistic effects on the inhibition of growth of P388 cells in vitro

McGown

Jayson²,

Pettit³

et al. 1998

Br J Cancer

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“…Protein estimation was done with Bradford reagent and the protein concentration was normalized. PKC activity was determined using the method of Yasuda et al 21 with minor modifications. 10 mg of the cell lysate was incubated for 20 min at 30°C in 40 ml of reaction containing 20 mM Tris-HCl (pH 7.5), 5 mM Mg(OAc)2, 0.2 mM CaCl2, 50 mM ATP, 2 mCi [g -32P ]ATP, 0.01 mg/ml Leupeptin and 50 mg/ml MBP4-14 as a substrate 18 with or without 25 mg/ml phosphatidylserine and 50 ng/ml TPA.…”

Section: Cell Lines B16-f10mentioning

confidence: 99%

Pentoxifylline modulates cell surface integrin expression and integrin mediated adhesion of B16F10 cells to extracellular matrix components

Ratheesh¹,

Ingle²,

Gude³

2007

Cancer Biology & Therapy

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Key woRdsPentoxifylline, adhesion, integrins, metastasis, B16-F10 melanoma, a5b1 integrin, Protein Kinase C AbbRevIAtIons AbstRActOur previous studies demonstrated that Pentoxifylline (PTX), a phosphodiesterase inhibitor, could inhibit the lung homing of B16-F10 melanoma cells in C57BL/6 mice. In this study we have looked at the effect of PTX on cell surface integrin expression and integrin mediated adhesion of B16-F10 melanoma cells. B16-F10 cells treated with PTX when injected through the tail vein of mice showed a 75% reduction in pulmonary nodules as compared to control untreated cells. PTX brought about a significant reduction in the integrin mediated adhesion of F10 cells to Fibronectin and Vitronectin (58.75% ± 3.4 S.E and 60% ± 1.7 S.E respectively if control was considered as 100%). This inhibition in adhesion was evident up to four hours only and treatment for 24 hours brought about an increase in adhesion (135.5% ± 0.5 S.E). Flow cytometric analysis showed higher surface expressions of av, a5 and aIIb integrin subunits in B16-F10 as compared to the low metastatic cell line B16-F1 suggesting a role for these integrins in determining the metastatic potential. PTX brought about a significant decrease in the cell surface expression of a5, aIIb and b1integrin subunits but not that of the av subunit on B16-F10 cells. PTX also brought about a reduction in the total cellular protein levels of b1 and av integrin subunits. Various isoforms of Protein Kinase C (PKC) has been shown to regulate integrin expression, localization and activity. Hence we looked at the effect of PTX on total cellular PKC activity. PTX brought about a significant reduction in total cellular PKC activity (82.66 ± 0.593). Collectively our results indicate that the antimetastatic action of PTX is mediated, at least in part through its effects on adhesion and the surface expression of specific integrin receptors.

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“…The measurement of PKC activity was performed as described by the manufacturer (GIBCO) using the protocol described by Thomas et al [27] and modified by Yasuda et al [28] which includes a pseudosubstrate inhibitor PKC (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). Membrane and cytosol fractions were separated by ultracentrifugation at 100 000 g for 60 min at 48C; the membrane fraction was then solubilized again with 1% (w/v) of Nonidet P-40 and recentrifuged at 100 000 g for 60 min at 48C.…”

Section: Determination Of Pkc Activitymentioning

confidence: 99%

Low density lipoprotein receptor expression and function in human polymorphonuclear leucocytes

Leclerc

Rivera

Perez-P

et al. 1997

Clinical and Experimental Immunology

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SUMMARYLow density lipoprotein receptors (LDLR), capable of internalizing LDL, are expressed in polymorphonuclear neutrophils (PMN). The expression was assessed using anti-LDLR antibody by flow cytometry. The internalization of LDL was assessed by: (i) quantification of the uptake of labelled LDL with 1,1 0 dicholoflurescein (DCF) by flow cytometry. This effect was not observed in monocytes or lymphocytes, and it was blocked by anti-LDLR antibody. The stimulation of LDL was optimal at 10 ¹ g of protein/ml. LDL was able to suppress DCF formation induced by phorbol myristate acetate (PMA) and PMA was unable to further stimulate LDL-treated cells, suggesting protein kinase-C (PKC) involvement in LDL effects. Using a PKC assay, LDL was shown to induce a two-fold increase in PKC translocation to the membrane. Thus, LDL increases PMN oxidative burst through a PKCdependent pathway.

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A synthetic peptide substrate for selective assay of protein kinase C

Cited by 293 publications

References 26 publications

Bryostatin 1-tamoxifen combinations show synergistic effects on the inhibition of growth of P388 cells in vitro

Bryostatin 1-tamoxifen combinations show synergistic effects on the inhibition of growth of P388 cells in vitro

Pentoxifylline modulates cell surface integrin expression and integrin mediated adhesion of B16F10 cells to extracellular matrix components

Low density lipoprotein receptor expression and function in human polymorphonuclear leucocytes

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