CYCLIC AMP-DEPENDENT PROTEIN KINASE STRUCTURE-FUNCTION

  • Maurer, Richard (PI)

Project: Research project

Project Details

Description

The proposed studies seek to examine the structural features of the
catalytic subunit of cAMP-dependent protein kinase which are necessary for
biological activity of the enzyme. In addition, the possible regulation of
the production of the enzyme will be examined. The principal approach will
involve analysis of catalytic subunit mutants to determine important
functional domains of the molecule. The isolation of mutants will be
facilitated by in vitro mutagenesis of catalytic subunit cDNA sequences.
The function of altered catalytic subunit cDNA sequences will be tested by
construction of expression vectors which will be introduced into mammalian
cells. Thus, the first specific aim of the proposed studies involves
isolation of catalytic subunit cDNA clones containing the complete coding
sequence. In preliminary studies a partial cDNA has been isolated and this
cDNA will be used as a hybridization probe to obtain a full-length clone.
The full-length clone will be inserted in an expression vector which will
be introduced into at least two cAMP-responsive cell lines for analysis of
the function of the cloned gene. After conditions for expression of the
cDNA clone have been determined, random mutations in the cDNA will be
introduced by treatment with bisulfite and mutants isolated and
characterized. Studies of genomic catalytic subunit sequences will also be
performed to determine if there are multiple copies of the gene.
Furthermore, analysis of the intron-exon organization of the gene may
provide some insight into functional domains of the enzyme. Finally, the
possible regulation of protein kinase gene expression will be examined by
hybridization analysis of mRNA levels in cAMP treated cells and tissues.
The overall goal of these studies is to increase the understanding of the
mechanisms which allow cAMP-dependent protein kinase to participate in
signal transduction and regulation of biological processes.
StatusFinished
Effective start/end date1/1/8612/31/88

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health

ASJC

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)

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