| Project/Area Number |
05454162
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
General medical chemistry
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| Research Institution | Yokohama City University |
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Principal Investigator |
OHNO Shigeo YCU Sch.Med.Dept.Mol.Bio.Professor, 医学部, 教授 (10142027)
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| Co-Investigator(Kenkyū-buntansha) |
OSADA Shinichi YCU Sch.Med.Dept.Mol.Bio.assistant Professor, 医学部, 助手 (00244484)
MIZUNO Keiko YCU Sch.Med.Dept.Mol.Bio.assistant Professor, 医学部, 助手 (90221803)
HIRAI Shuichi YCU Sch.Med.Dept.Mol.Bio.lecturer, 医学部, 講師 (80228759)
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| Project Period (FY) |
1993 – 1994
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| Project Status |
Completed (Fiscal Year 1994)
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| Budget Amount *help |
¥7,800,000 (Direct Cost: ¥7,800,000)
Fiscal Year 1994: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 1993: ¥5,000,000 (Direct Cost: ¥5,000,000)
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| Keywords | protein kinase C / phospholipids / diacylglyverol / tumor promoters / PKC substrates / PKC binding proteins / TPA / TPA-response element |
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| Research Abstract |
Tumor promoting phorbol esters such as TPA/PMA activates transcription of a variety of genes through TPA-response elements. We found that the overexpression of kinase-knockout protein kinase C (PKC) inhibit TRE-CAT expression, suggesting the presence of a protein which bind to PCK and transmit signals downstream. On the other hand, we also found that MARCKS,a PKC substrate, shows very high affinity to PKC with the sacrifice of the efficiency of phosphorylation by PKC.In fact, MARCKS binds tightly to PKC.These results prompted us to identify PKC binding proteins which might be the substrate of PKC.For this purpose, we screened NIH3T3 cDNA expression library by using labeled-PKCdelta as a probe. We have isolated more than ten independent cDNA clones where MARCKS was included. The rest of the cDNA clones encoded novel proteins with various structural motifs. Experiments directed to compare the binding efficiency of these PKC binding proteins with PKC family members revealed that most of them can bind and be phosphorylated by cPKCalpha, nPKCdelta, nPKCepsilon, aPKClambda, albeit to different degrees depending on the PKC isotypes in vitro.
These results indicate that we can identify novel PKC substrates using the strategy used in the present study. Further, there are differences in the affinity of PKC substrates/binding proteins to different PKC isotypes in vitro, suggesting the presence of PKC type-specific substrates in vivo. Further experiments directed for the identification of the in vivo function of the PKC type-specific substrates/binding proteins will provide critical tools to clarify the role of respective PKC members in intact cells.
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