Protein phosphorylation in cultured endothelial cells

Mackie, Ken; Lai, Yvonne; Nairn, Angus C.; Greengard, Paul; Pitt, Bruce R.; Lazo, John S.

doi:10.1002/jcp.1041280304

Cited by 56 publications

(28 citation statements)

References 75 publications

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“…The 4.0-4.7) and, when subjected to limited proteolysis with S. aureus V8 protease, generated the same phosphopeptide map as the bovine protein. The 87-kDa protein has been identified by similar techniques in bovine vascular endothelial cells (20) and rat heart (unpublished results). Therefore, within a species the 87-kDa protein from each tissue was identical, although the concentration varied among the tissues, and the protein from rat tissues was of lower molecular mass than the protein from bovine tissues.…”

Section: Resultsmentioning

confidence: 70%

Widespread occurrence of "87 kDa," a major specific substrate for protein kinase C.

Albert¹,

Walaas²,

Wang³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

162

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An 87-kDa phosphoprotein, identified previously as a major, specific substrate for Ca2+/phospholipid/diacylglycerol-dependent protein kinase (protein kinase C) in broken cell preparations from rat brain, has been characterized with respect to its species, tissue, and subcellular distribution. A similar protein was present in monkey, human, mouse, and bovine brain and in Torpedo californica electric organ. The protein was also identified in a variety of nonneuronal rat and bovine tissues. The rat protein had an apparent molecular mass 4-7 kDa lower, and was slightly more acidic, than the protein in bovine tissues. The 87-kDa proteins from various bovine tissues were identical by the following criteria: each was phosphorylated by exogenous protein kinase C, was ofcomparable molecular mass, generated multiple spots within the pH range of 4.4-4.9 upon isoelectric focusing, yielded identical patterns upon digestion with Staphylococcus aureus V8 protease, and was recognized by a specific 87-kDa antiserum. The relative concentrations of the 87-kDa protein in bovine tissues were highest in brain, spleen, and lung, moderate in testis, pancreas, adrenal, kidney, and liver, and lowest in heart and skeletal muscle. In the brain, the 87-kDa protein was concentrated in the synaptosomal membrane and in the cytosol. The membrane-bound protein was extractable with nonionic detergents but not with NaCI. This species, tissue, and subcellular distribution of the 87-kDa protein is similar to that of protein kinase C.

show abstract

Section: Resultsmentioning

confidence: 70%

Widespread occurrence of "87 kDa," a major specific substrate for protein kinase C.

Albert¹,

Walaas²,

Wang³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

162

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“…We have identified the 100-kDa protein in cultured epithelial cells (41), endothelial cells (42), parotid gland, testis, and vas deferens (unpublished results). It is also likely that the 100-kDa protein is identical to phosphorylated proteins of similar molecular weight observed in previous studies of endogenous CaM-dependent protein phosphorylation in pancreas and in a number ofother mammalian tissues.…”

Section: )mentioning

confidence: 83%

Identification of calmodulin-dependent protein kinase III and its major Mr 100,000 substrate in mammalian tissues.

Nairn¹,

Bhagat²,

Palfrey³

1985

Proc. Natl. Acad. Sci. U.S.A.

199

125

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A major substrate, Mr 100,00 (100 kDa), for a Ca2+/calmodulin (CaM)-dependent protein kinase found in many mammalian tissues has been purified from rat pancreas. The purified substrate was used to identify and partially purify a CaM-dependent protein kinase (CaM kinase Il) from rat pancreas. The physical properties and substrate specificity of CaM kinase HI were distinct from those of all known CaMdependent protein kinases. Only CaM kinase m was able to phosphorylate the 100-kDa protein; synapsin I, phosphorylase b, myosin light chain, and histone were poor substrates for this enzyme. Polyclonal antibodies, raised against the purified 100-kDa protein, recognized the protein in a variety of mammalian tissues and cell lines. Immunoassay revealed that the 100-kDa protein made up 0.3-1.7% of the total cytosolic protein in these samples. Analysis of CaM kinase m revealed that the enzyme had a similar widespread tissue distribution.

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“…Receptor phos phorylation by calcium-dependent kinases inactivates some receptors until calcium is removed, reducing kinase activity and allowing phosphatases to dephosphorylate the receptor [33][34][35][36]. Directly related to such a mecha nism would be Rout, the efflux rate of cytosolic calcium, either out of the cell or to membrane storage pools, and its relationship to Rjn.…”

Section: Discussionmentioning

confidence: 99%

Mechanisms of Flow-Mediated Signal Transduction in Endothelial Cells: Kinetics of ATP Surface Concentrations

1992

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Intracellular free calcium ([Ca²⁺]_i) was measured in single cells of a confluent endothelial monolayer subjected to defined flow. Flow medium containing adenosine triphosphate (ATP) was used to study the influence of flow forces upon agonist-response coupling as mediated via the P2_y-purinoceptor. [Ca²⁺]_i responses were highly sensitive to the fluid motion at the cell surface; consecutive small increases of flow stimulated large [Ca²⁺]i transients with the levels returning to baseline at the new flow rate within 250 s. The characteristics of [Ca²⁺]_i transients were also influenced by decreasing flow. Since potent ecto-nucleotidases at the endothelial cell surface rapidly degrade ATP, we postulated that a combination of flow and degradative enzymes regulates the mass transport of ATP in the boundary layer. The hypothesis predicts that step increases of flow exceed the capacity of the ectonucleotidases and allow ATP to reach the receptor. Experiments were conducted to compare ATP and ADP_βS, a nonhydrolyzable ATP analog that resists degradation by surface ectonucleotidases, and calculations of ATP mass transport to the cell surface were compared to estimates of surface clearance rates. Calculations of mass transport coefficients for ATP in the boundary layer demonstrated that changes of flow which elicited a prominent [Ca²⁺]_i response represented 26-73 % changes in the mass transport of ATP from the bulk fluid. When steady-state mass transport coefficients for ATP under various flow conditions were compared with the estimated rate constant for surface degradation of ATP, ratios close to unity were obtained. These results suggest that both boundary layer mass transport and ATP clearance rates can be rate-limiting for flow-mediated activation of the P_2y-receptor. The experiments provide evidence for differential signal transduction responses in the endothelium driven by diffusion gradients (derived from both the blood and the vessel wall), which are likely to vary widely in the complex flow fields encountered in vivo.

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Protein phosphorylation in cultured endothelial cells

Cited by 56 publications

References 75 publications

Widespread occurrence of "87 kDa," a major specific substrate for protein kinase C.

Widespread occurrence of "87 kDa," a major specific substrate for protein kinase C.

Identification of calmodulin-dependent protein kinase III and its major Mr 100,000 substrate in mammalian tissues.

Mechanisms of Flow-Mediated Signal Transduction in Endothelial Cells: Kinetics of ATP Surface Concentrations

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