Project: Research project

Project Details


The purpose of this proposal is to determine the role of cAMP-
dependent protein phosphorylation in C1 secretion across tracheal
epithelia. Using cultured tracheal epithelial cells, proteins will be
phosphorylated by an hour's incubation with 32PO4. Cytosolic and
membrane proteins will be separated. The time course of changes
in phosphorylation of individual proteins in response to agents
which increase intracellular cAMP will be followed, and compared
to the time course of change in Cl secretion. The catalytic
subunit of cAMP-dependent protein kinase, AT32P and cAMP will
be introduced into apical membrane vesicles. Changes in
phosphorylation will be correlated with changes in Cl influx
measured with the C1-sensitive fluorescent probe, SPQ. Apical
membranes will be used because they are relatively enriched in
membrane proteins. To this same end, we will try and repeat
these studies on vesicles from which cytoskeletal elements have
been removed. Phosphorylation changes in normal cultured cells
and vesicles will be compared with those of CF cells. Levels of
cAMP-dependent protein kinase and its sensitivity to cAMP will
be compared in normal and CF cells. Endogenous substrates will
be measured in cell extracts. The various form of the RI and RII
regulatory subunits of cAMP-dependent protein kinase will be
covalently labelled with a photo-affinity derivative of cAMP, and
their levels determined following separation on two-D gels. All
experiments will be performed initially on dog or cow cells. Once
the techniques are established, non-CF and CF human cells will be
compared. Because, CF may involve a defective interaction
between Ca- and cAMP-dependent systems, similar experiments
to those on cAMP-dependent protein kinase and phosphorylation
will be performed on the calmodulin-dependent protein kinase and
protein kinase C. We hope to obtain information on how normal
C1 secretion is regulated. Membrane proteins which change their
phosphorylation as rapidly as the change in C1 secretion are
candidates for the apical membrane C1 channel. Differences in
kinase levels or phosphorylation patterns in CF would provide
information as to the basic metabolic defect in this disease.
Effective start/end date3/1/882/28/91


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


  • Medicine(all)


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