Cyclic 3′,5′-Adenosine Monophosphate-Dependent Kinase and Phosphoproteins in Human Amnion 1

Abstract

3′,5′-Cyclic adenosine monophosphate (cAMP) modulates prostaglandin production in human amnion membranes. The major effects of cAMP are presumably mediated through the phosphorylation of specific regulatory phosphoproteins following cAMP activation of cAMP-dependent protein kinase. Cyclic AMP-dependent protein kinase and phosphoproteins have not previously been characterized in human amnion. Total homogenates, cytosol, and membrane fractions from human amnion were examined for [³H]cAMP binding activity and cAMP-dependent kinase activity. cAMP-dependent kinase activity was barely detectable in crude amnion fractions. Cytosol was therefore partially purified by DEAE column chromatography for further examination. Two peaks of coincident [³H]cAMP binding and cAMP-dependent kinase activity were demonstrated at 70 and 140 mM NaCl, characteristic of the Type I and Type II cAMP-dependent protein kinase isozymes. [³H]cAMP binding to the material from both peak fractions was saturable and reversible. Scatchard analysis of [³H]cAMP binding to the peak fractions was linear for peak I and curvilinear for peak II. Assuming a one-site model, [³H]cAMP binding to the Type I isozyme showed a K_D = 4.17 × 10^-8 M and B _max = 73 pmole/mg protein; using a two-site model, [³H]cAMP binding to the high-affinity site for the Type II isozyme had a K_D = 3.94 × 10^-8 M and B _max = 6.3 pmole/mg protein. Other cyclic nucleotides competed for these [³H]cAMP binding sites with a potency order of cAMP > cGMP > (BU)₂cAMP. cAMP caused a dose-dependent increase in cAMP-dependent kinase activity in the peak fractions; half-maximal activation was observed with 5.0 × 10^-8 M cAMP. The ability of cAMP to increase phosphorylation of endogenous proteins in both crude amnion cytosol and cytosol from cultures of amnion epithelial cells was assessed using [³²P]ATP, SDS-polyacrylamide gel electrophoresis and autoradiography. cAMP stimulated ³²P incorporation into three proteins having M _r = 80,000, 54,000, and 43,000 (P < .01). Half-maximal ³²P incorporation into these proteins occurred at 1.0 × 10^-7 M cAMP. cAMP-dependent kinase is present in human amnion; specific cAMP-enhanced phosphoproteins are also present. Hormones elevating cAMP levels in amnion may exert their effects by activating cAMP-dependent kinase and phosphorylating these phosphoproteins.