Abstract
We have engineered an acrylodan-modified derivative of the catalytic subunit of cyclic AMP-dependent protein kinase (cAPK) whose fluorescence emission signal has allowed the synergistic binding between nucleotides and physiological inhibitors of cAPK to be examined (Whitehouse, S., and Walsh, D. A. (1983) J. Biol. Chem. 258, 3682-3692). In the presence of the regulatory subunit, RI, the affinity of cAPK for adenosine, ADP, AMPPNP (adenosine 5'-(beta, gamma-imino)triphosphate), or ATP was 5-, 50-, 120-, and 15,000-fold enhanced, while in the presence of the heat-stable inhibitor protein of cAPK (PKI), there was a 3-, 20-, 33-, and 2000-fold enhancement in the binding of these nucleotides, respectively. A short inhibitor peptide, PKI-(14-22), enhanced the binding of ADP to the same degree as did full-length PKI (20-fold) but, in contrast, did not significantly enhance the binding of ATP or AMPPNP. The full binding synergism between PKI and either ATP (2000-fold) or AMPPNP (33-fold) to cAPK could, however, be mimicked by a longer peptide, PKI-(5-24), suggesting that the PKI NH2 terminus (residues 5-13) is most likely critical. Since this region is remote from the ATP gamma-phosphate, the binding synergism must arise through an extended network communication mechanism between the PKI NH2 terminus and the ATP binding site.
Highlights
Protein kinases catalyze the transfer of phosphate from ATP to protein substrates and are central regulatory elements of all known pathways of signal transduction
The recent elucidation of two x-ray crystal structures containing the cyclic AMP-dependent protein kinase (cAPK) catalytic subunit, a 20-residue inhibitor peptide (PKI-(5–24)) and either MnATP [17] or MnAMPPNP [18] reveal the apparent structural basis for the binding synergism, which is attributed to an observed network of hydrogen bonds directly interlinking the nucleotide ␥-phosphate, the enzyme’s glycine-rich loop, and the P-site Ala of the inhibitor peptide
We have generated a simple and sensitive fluorescence technique for measuring nucleotide binding to cAPK by engineering an acrylodan-labeled derivative of the catalytic subunit (AcrcAPK)
Summary
Protein kinases catalyze the transfer of phosphate from ATP to protein substrates and are central regulatory elements of all known pathways of signal transduction. CAPK exists as an inactive tetrameric holoenzyme which, upon binding cAMP, undergoes dissociation to a single dimeric regulatory subunit and two free, active catalytic subunits [8]. The recent elucidation of two x-ray crystal structures containing the cAPK catalytic subunit, a 20-residue inhibitor peptide (PKI-(5–24)) and either MnATP [17] or MnAMPPNP [18] reveal the apparent structural basis for the binding synergism, which is attributed to an observed network of hydrogen bonds directly interlinking the nucleotide ␥-phosphate, the enzyme’s glycine-rich loop, and the P-site Ala of the inhibitor peptide. The principal limitation in many cases, is the lack of a fluorescent signal upon
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