Substrate Enhances the Sensitivity of Type I Protein Kinase A to cAMP

Abstract

The functional significance of the presence of two major (types I and II) isoforms of the cAMP-dependent protein kinase (PKA) is still enigmatic. The present study showed that peptide substrate enhanced the activation of PKA type I at low, physiologically relevant concentrations of cAMP through competitive displacement of the regulatory RI subunit. The effect was similar whether the substrate was a short peptide or the physiological 60-kDa protein tyrosine hydroxylase. In contrast, substrate failed to affect the cAMP-sensitivity of PKA type II. Size exclusion chromatography confirmed that substrate acted to physically enhance the dissociation of the RI␣ and C␣ subunits of PKA type I, but not the RII␣ and C␣ subunits of PKA type II. Substrate availability can therefore fine-tune the activation of PKA type I by cAMP, but not PKA type II. The cAMP-dissociated RII and C subunits of PKA type II reassociated much faster than the PKA type I subunits in the presence of substrate peptide. This suggests that only PKA type II is able to rapidly reverse its activation after a burst of cAMP when exposed to high substrate concentration. We propose this as a possible reason why PKA type II is preferentially found in complexes with substrates undergoing rapid phosphorylation cycles.The cAMP-dependent protein kinase (PKA) 1 is a model for protein kinases because of its universal distribution and its relative simplicity because the catalytic moiety and the autoinhibitory moiety are on different subunits. The mammalian tetrameric PKA consists of two catalytic (C) subunit monomers and a regulatory (R) subunit dimmer. The binding of two molecules of cAMP to each R subunit favors dissociation of the C and R subunits. The kinase exists in two major isoforms with different R subunits, RI and RII, each with ␣-and ␤-subforms. The biological significance of the presence of two major isozymes is still uncertain. It was early noticed that RI predominated in many cells with rapid proliferation, rapid growth in cell size, or malignantly transformed cells (1-4). More recently it has been shown by homologous recombination that RI␣Ϫ/Ϫ mice die in embryonic life, whereas RII␤Ϫ/Ϫ, RII␣Ϫ/Ϫ, and RI␤Ϫ/Ϫ mice have less obvious defects, mainly in differentiation of adipose tissue (RII␤) and neural functions (RI␤) (5). An important difference between RI and RII is their relative affinity for anchoring proteins that confine PKA type II, and in some cases PKA type I, to subcellular compartments (see Refs. 6 and 7 for recent reviews). Another striking difference is the ability of PKA type II, but not type I, to become autophosphorylated (8 -10). The interface between the R and C subunits of PKA is complex. The RI and RII subunits bind in part to non-overlapping areas of the C subunit (11). The R subunits contact the substrate binding site of the C subunit as well as areas distant from this site (12) and are, through their (pseudo)substrate moiety, believed to displace substrate from the C subunit and thereby inhibit the kinase activity (10, 12). The subs...

Regulation of cAMP-dependent Protein Kinases

“…Binding of RI to the C subunits was reduced by ϳ50% upon treatment with 50 M forskolin and 500 M IBMX, which we used to get to a sustained increase of [cAMP] i , in agreement with previous results (Fig. 1 (35,36). In contrast, the PrKY/RI␤ interaction was not influenced by [cAMP] i (Fig.…”

Characterization of Substrates That Have a Differential Effect on Saccharomyces cerevisiae Protein Kinase A Holoenzyme Activation

“…It has been demonstrated that both the mammalian type I␣ and the type II␤ holoenzymes are more stable in the presence of cAMP than thought previously and that the substrate can enhance the dissociation of PKA type I at low, physiologically relevant concentrations of cAMP but failed to affect the interaction of the subunits of type II kinase (26,27). In our laboratory, we have demonstrated the participation of the peptide substrate in the activation of a fungal (Mucor rouxii) holoenzyme by cAMP (29).…”

“…It has been demonstrated recently that although the addition of a molar excess of cAMP to the type I␣ PKA holoenzyme causes partial dissociation, it is only upon addition of a PKA peptide substrate, together with cAMP that full dissociation occurs. The authors also demonstrate that substrate plays a differential role in the activation of type I versus type II holoenzymes, which could explain some important functional differences between PKA isoforms (27,28). We have shown, some years ago, that peptide substrates were involved in the activation of a fungal PKA by cAMP (29).…”

Cyclic GMP from the surrounding somatic cells regulates cyclic AMP and meiosis in the mouse oocyte

“…PKAII is half-maximally activated at ~500 nM cAMP (Dostmann and Taylor, 1991;Viste et al, 2005), and based on data in these publications a decrease in cAMP from ~660 to ~140 nM would reduce the activity of PKAII by 90%. PKAI activity would also decrease, although the extent of this decrease is unknown, as values for the cAMP concentrations that half-maximally activate PKAI vary from 70 to 500 nM depending on the type and concentration of the substrate (Viste et al, 2005).…”

“…However, the magnitude of these changes was difficult to interpret because the oocytes were removed from their follicles, which would decrease cAMP, and because phosphodiesterase inhibitors were present, which would increase cAMP. Thus, it is unknown whether the decreases in cAMP that were observed in response to LH receptor stimulation were sufficient to affect the activity of PKA, which requires cAMP in the range of 70-500 nM for half-maximal activity (Dostmann and Taylor, 1991;Viste et al, 2005). LHstimulated decreases in cGMP occur in oocytes and whole follicles from hamsters (Hubbard, 1986), but whether the magnitude of these changes is physiologically significant and whether these decreases occur in other species are unknown.…”