Characterization of the Catalytic and Nucleotide Binding Properties of the α-Kinase Domain of Dictyostelium Myosin-II Heavy Chain Kinase A

Abstract

The α-kinases are a widely expressed family of serine/threonine protein kinases that exhibit no sequence identity with conventional eukaryotic protein kinases. In this report, we provide new information on the catalytic properties of the α-kinase domain of Dictyostelium myosin-II heavy chain kinase-A (termed A-CAT). Crystallization of A-CAT in the presence of MgATP yielded structures with AMP or adenosine in the catalytic cleft together with a phosphorylated Asp-766 residue. The results show that the β- and α-phosphoryl groups are transferred either directly or indirectly to the catalytically essential Asp-766. Biochemical assays confirmed that A-CAT hydrolyzed ATP, ADP, and AMP with kcat values of 1.9, 0.6, and 0.32 min(-1), respectively, and showed that A-CAT can use ADP to phosphorylate peptides and proteins. Binding assays using fluorescent 2'/3'-O-(N-methylanthraniloyl) analogs of ATP and ADP yielded Kd values for ATP, ADP, AMP, and adenosine of 20 ± 3, 60 ± 20, 160 ± 60, and 45 ± 15 μM, respectively. Site-directed mutagenesis showed that Glu-713, Leu-716, and Lys-645, all of which interact with the adenine base, were critical for nucleotide binding. Mutation of the highly conserved Gln-758, which chelates a nucleotide-associated Mg(2+) ion, eliminated catalytic activity, whereas loss of the highly conserved Lys-722 and Arg-592 decreased kcat values for kinase and ATPase activities by 3-6-fold. Mutation of Asp-663 impaired kinase activity to a much greater extent than ATPase, indicating a specific role in peptide substrate binding, whereas mutation of Gln-768 doubled ATPase activity, suggesting that it may act to exclude water from the active site.

Keywords: adenosine; aspartyl phosphate; atypical protein kinase; catalysis; enzyme kinetics; myosin-II heavy chain kinase; protein kinase; x-ray crystallography; α-kinase.

FIGURE 1.

Structure of A-CAT bound to adenosine or AMP. A, schematic representation of A-CAT bound to adenosine (A-CAT·ADN·P) with the N-terminal lobe (residues 552–655) colored salmon, the central core (residues 656–712) colored cyan, and the C-terminal lobe and tail (residues 713–842) colored blue. The N and C termini, β-sheets, and α-helices are labeled. Phosphate (Pi1), Asp(P)-766 (P-D766), and adenosine (ADN) are shown as sticks with the adenosine carbon atoms colored yellow. The zinc atom is shown as a magenta sphere. B and C, close-up views showing the catalytic cleft of A-CAT·ADN·P and A-CAT·AMP·P. Adenosine, AMP, Pi1, and Asp(P)-766 are shown as sticks. The mesh shows the 2F_o − F_c electron density map contoured at the 2σ level. D, alignment of the A-CAT·ADN·P (gray), A-CAT·AMP·P (salmon), and A-CAT·ADP·P (blue) structures shows that there is little or no change in the positions of the P-loop, N/D-loop, or key catalytic residues. E, view of the A-CAT·ADN·P active site showing the interactions (dashed lines) made by Asp(P)-766 and Pi1.

FIGURE 2.

A-CAT can hydrolyze ATP, ADP, and AMP and is able to use ADP to phosphorylate peptide and protein substrates. A, hydrolysis of ATP (▴), ADP (○), and AMP (●) by A-CAT. The initial rate of P_i generation at each nucleotide concentration was measured as described under “Experimental Procedures.” Kinetic constants, obtained by fitting the data to a hyperbolic curve, are listed in Table 3. B, Phos-tag gel analysis of MBP phosphorylation. Kinase assays were carried out using A-CAT and MBP in the absence of nucleotide or with 0.5 mm ATP or 0.5 mm ADP as described under “Experimental Procedures.” Samples were taken at the indicated times (ON = 12 h) and electrophoresed on SDS gels polymerized in the presence (+) or absence (−) of the Phos-tag molecule. The gels were stained with Coomassie Blue to visualize proteins. Phosphorylated forms of MBP (P-MBP) electrophorese with a reduced mobility in the presence of the Phos-tag molecule. C and D, mass spectrometry analysis of the YAYDTRYRR peptide incubated with ADP and kinase-dead A-CAT-D766S (C) or A-CAT (D). The full MS spectra of [M + 2H]²⁺ in the positive mode is shown. A peak corresponding to the unphosphorylated peptide (m/z = 652.83) was detected in the sample incubated with A-CAT-D766S. An abundant peak corresponding to the phosphorylated peptide (m/z = 692.81) was detected in the sample incubated with A-CAT. Kinase assays were performed as described under “Experimental Procedures.”

FIGURE 3.

Binding of mant-ADP and mant-ATP to A-CAT. A, fluorescence emission spectra obtained upon excitation at 280 nm were recorded for 28 μm mant-ATP (dashed line), 2 μm A-CAT (dotted line), and a mixture of 28 μm mant-ATP and 2 μm A-CAT (black line). Assays contained 2 mm MgCl₂ and were carried out as described under “Experimental Procedures.” In the presence of A-CAT, there is a large increase in the fluorescence emission of mant-ATP in the 400–500 nm region. B and C, change in fluorescence emission at 430 nm (ΔF) was measured as mant-ATP (B) or mant-ADP (C) and titrated into a solution containing 2 μm A-CAT in the presence (●) or absence (○) of 2 mm MgCl₂. The lines show the best fit of the data to a hyperbolic binding curve. D, change in fluorescence emission at 430 nm (ΔF) was measured as mant-ADP was titrated into solutions containing 2 μm A-CAT and 2 mm MgCl₂ in the presence of 9, 12, 40, and 80 μm ATP. Decreased ΔF values reflect competition between ATP and mant-ADP for binding to A-CAT. The binding constants determined by fitting the data in B–D are listed in Table 4.

FIGURE 4.

Schematic diagram showing A-CAT interactions with ATP and Mg²⁺ ions. Residues in A-CAT that interact with ATP, Mg1, and Mg2 (magenta spheres) and that were examined in this study by site-directed mutagenesis are shown as sticks. Acidic residues are colored blue, and basic residues salmon and other residues are gray.

FIGURE 5.

Kinase and ATPase activities of wild-type and mutant A-CAT. A, kinase and ATPase activity of wild-type (WT) A-CAT and the indicated mutants were assayed as described under “Experimental Procedures.” B, ATPase and kinase activities for the WT A-CAT and the R592L and K722N mutants were assayed at varying ATP concentrations. The kinetic constants obtained by fitting the data to a hyperbolic curve are listed in Table 3.

FIGURE 6.

Comparison of the A-CAT and PKA active sites. Active site residues in A-CAT (Protein Data Bank code 3LKM) (gray) and PKA (Protein Data Bank code 1ATP) (blue) were aligned using PyMOL. The A-CAT structure contains AMP, and the PKA structure contains ATP.