PROTEIN KINASE II AND PROTEIN PHOSPHATASE IN CALCIUM STIMULATED GENE EXPRESSION

钙刺激基因表达中的蛋白激酶 II 和蛋白磷酸酶

• 批准号: 5210700
• 负责人: THOMAS R SODERLING
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5210700
• 关键词: calcium; calmodulin dependent protein kinase; enzyme inhibitors; gene expression; genetic regulatory element; human genetic material tag; microinjections; phosphoprotein phosphatase; thapsigargin; tissue /cell culture; transfection

This project employs a multifaceted approach to assess the roles of calcium/calmodulin-dependent protein kinase II (CaM-kinase II) and protein phosphatases in the regulation of Ca2+-stimulated gene transcription in PC12 cells. We will focus on several genes which utilize the enhancer protein CREB and/or CRE-related elements as found in the 5'-flanking sequences of c-fos, nur/77 (NGF1-B), and tyrosine hydroxylase genes. Ca2+-dependent transcription of the three endogenous genes will be initiated by three "Ca2+ agonists": depolarization (KC1 plus BAY K8644), ionomycin or thapsigargin. Initial experiments will determine the effect on MRNA levels of cell-permeable inhibitors of CaM-kinase II (KN-62) or protein phosphatases (okadaic acid) or transfection with a dominant negative mutant of CREB. PC12 cells will also be lipofected with CRE- or CaRE-CAT constructs, and the same paradigms used to assess CAT expression. Cells will also be lipofected with a constitutively-active mutant of CaM- kinase II which should give Ca2+-independent gene expression. Synthetic peptide inhibitors of several protein kinases and phosphatases will be used as probes by microinjection into PC12 cell stably-transfected with a CRE- or CaRE-lacZ reporter gene, and beta-galactosidase expression in single cells determined in response to the "Ca2+-agonists." The second major focus of the project will examine phosphorylation of CREB and delta-CREB, which lacks residues 88-102, by CaM-kinase II. Particular attention will be given to phosphorylation of Ser(133) and Ser(96) (lacking in delta-CREB), a consensus site for CaM-kinase II. In addition to kinetic characterization (Km and Vmax) of these substrates for CaM-kinase II, the (32)p-labeled CREB will be tested as substrate for several protein phosphatases. CREB and delta-CREB, thiophosphorylated on Ser (133) and/or Ser(98), will be microinjected into the cytoplasm or nucleus of CaRE-lacZ transfected PC12 cells, and beta-galactosidase expression determined. Lastly, teratocarcinoma F9 cells will be transfected with CREB or delta- CREB and the constitutively-active CaM-kinase II mutant to determine CAT expression. These complementary approaches, deemed necessary because of the "crosstalk" between the multifunctional protein kinases and the degeneracy in the enhancer binding elements of transcriptionally-regulated genes, should allow us to dissect the roles of CaM-kinase II, protein phosphatases, and CREB in calcium-stimulated gene transcription.

本项目采用多方面的方法来评估