THROMBIN AND HISTAMINE RAPIDLY STIMULATE THE PHOSPHORYLATION OF THE MYRISTOYLATED ALANINE-RICH C-KINASE SUBSTRATE IN HUMAN UMBILICAL VEIN ENDOTHELIAL-CELLS - EVIDENCE FOR DISTINCT PATTERNS OF PROTEIN-KINASE ACTIVATION

被引:17
作者
JACOBSON, BC
POBER, JS
FENTON, JW
EWENSTEIN, BM
机构
[1] BRIGHAM & WOMENS HOSP,DIV HEMATOL ONCOL,BOSTON,MA 02115
[2] BRIGHAM & WOMENS HOSP,DEPT PATHOL,BOSTON,MA 02115
[3] HARVARD UNIV,SCH MED,DEPT MED,BOSTON,MA 02115
[4] HARVARD UNIV,SCH MED,DEPT PATHOL,BOSTON,MA 02115
[5] NEW YORK DEPT HLTH,WADSWORTH CTR LABS & RES,ALBANY,NY 12201
[6] UNION UNIV,DEPT PHYSIOL & CELL BIOL,ALBANY,NY 12208
[7] UNION UNIV,DEPT BIOCHEM,ALBANY,NY 12208
关键词
D O I
10.1002/jcp.1041520121
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
Human alpha-thrombin and histamine each stimulates protein phosphorylation in human umbilical vein endothelial cells (HUVEC). We have identified the most prominent of these phosphoproteins by immunoprecipitation as the human homolog of the widely distributed myristoylated alanine-rich C-kinase substrate (MARCKS). Stimulation by 0.1-10 U/ml of alpha-thrombin produces a time-dependent, sustained (plateau 3-5 min) level of MARCKS phosphorylation. MARCKS phosphorylation requires thrombin catalytic activity but not receptor binding and is also seen in response to stimulation by a peptide, TR (42-55), that duplicates a portion of the thrombin receptor tethered ligand created by thrombin proteolytic activity. One micromolar histamine, like alpha-thrombin, produces sustained phosphorylation of MARCKS (plateau 3-5 min). In contrast, 100-mu-M histamine results in rapid but transient MARCKS phosphorylation (peak 1-3 min). HUVEC treated with 100-mu-M histamine for 5 min can be restimulated by alpha-thrombin but not fresh histamine, suggesting that the histamine receptor was desensitized. MARCKS phosphorylation can also be induced by several exogenous protein kinase C (PKC) activators and both alpha-thrombin- and histamine-induced MARCKS phosphorylation are inhibited by the PKC antagonist staurosporine. However, while prolonged PMA pretreatment ablates histamine-induced MARCKS phosphorylation, the ability of thrombin to induce MARCKS phosphorylation is retained. These findings provide evidence for agonist-specific pathways of protein kinase activation in response to thrombin and histamine in HUVEC.
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页码:166 / 176
页数:11
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