| Budget Amount *help |
¥6,200,000 (Direct Cost: ¥6,200,000)
Fiscal Year 1991: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 1990: ¥3,600,000 (Direct Cost: ¥3,600,000)
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| Research Abstract |
The cANP response element(CRE)is an inducible enhancer of genes such as those of preproenkephaun and somatostatin, with can be transcribed in response to increased cAMP levels. We previously identified the CRE-binding protein, CRE-BP1, by CDNA cloning. CRE-BPI has a "B-ZIP" structure as a DNA-binidng domain and binds to CRE as a homodimer or a heterodimer with c-Jun. We have investigated the phosphorylation of CRE-BP1. We also analyzed the functional domains of CRE-BPL to examine the role of protein containing the phosphorylation sites. The human recombinant CRE-BPl was phosphorylated by cAMP-dependent protein Idnase ard protein kinase C, in vitro. These two protein kinases modified distinct semine residues of CRE-BP1. Ser-62 was the phosphorylation site by cAMP-dependent protein kinase, whereas two semine residues, Ser-340 and Ser-367 were the major protein kinase C phophorylation sites. By examining the activity of a series of deletion and point mutants of CRE-BP1, two functional dom
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ains of CRE-BPL were identified : the putative metal finger structure in the amino-terininal region and the leucine zipper motif linked to a cluster of basic amino acids in the carboxyl-terminal region. The former was a transcriptional activation domain, and the latter was a DNA-binding domain. Therefore, Ser-62 which is phosphorylated by cAMP-dependent protein kinase is close to the transcriptional activation domain, whereas Ser-340 and Ser-367, the protein Idnase C phosphorylation sites, are located in the basic region of the DNA-binding domain. These results suggest that transcriptional and DNA-binding activities of CRE-BPL are regulated by phosphorylation with these protein kinases.
We have also examined the effect of phosphorylation by CK-H on the activity of c-myb oncogene product(c-Myb). c-Myb is a transcriptional activator that can bind to the specific DNA sequence. Casein kinase H(CK-II)is known to phosphorylate Ser- 1 1 and Ser- 12 of c-Myb. To examine the effect of phosphorylation of these Ser residues on c-Myb activity, we replaced these Ser residues to alanine. Alanine mutant had about 2-fold higher transactivating capacity than the normal c-Myb. These results indicate that phophorylation of c-Myb by CK-H represses the c-Myb activity. Since CK-H activity is liked to the cellular growth control, these results may give a clue to understand a linkage between transcriptional factors and cellular growth control. Less
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