Autophosphorylation Activates Dictyostelium Myosin II Heavy Chain Kinase A by Providing a Ligand for an Allosteric Binding Site in the α-Kinase Domain

Abstract

Dictyostelium discoideum myosin II heavy chain kinase A (MHCK A), a member of the atypical ␣-kinase family, phosphorylates sites in the myosin II tail that block filament assembly. Here we show that the catalytic activity of A-CAT, the ␣-kinase domain of MHCK A (residues 552-841), is severely inhibited by the removal of a disordered C-terminal tail sequence (C-tail; residues 806 -841). The key residue in the C-tail was identified as Thr 825 , which was found to be constitutively autophosphorylated. Dephosphorylation of Thr 825 using shrimp alkaline phosphatase decreased A-CAT activity. The activity of a truncated A-CAT lacking Thr 825 could be rescued by P i , phosphothreonine, and a phosphorylated peptide, but not by threonine, glutamic acid, aspartic acid, or an unphosphorylated peptide. These results focused attention on a P i -binding pocket located in the C-terminal lobe of A-CAT. Mutational analysis demonstrated that the P ipocket was essential for A-CAT activity. Based on these results, it is proposed that autophosphorylation of Thr 825 activates ACAT by providing a covalently tethered ligand for the P i -pocket. Ab initio modeling studies using the Rosetta FloppyTail and FlexPepDock protocols showed that it is feasible for the phosphorylated Thr 825 to dock intramolecularly into the P i -pocket. Allosteric activation is predicted to involve a conformational change in Arg 734, which bridges the bound P i to Asp 762 in a key active site loop. Sequence alignments indicate that a comparable regulatory mechanism is likely to be conserved in Dictyostelium MHCK B-D and metazoan eukaryotic elongation factor-2 kinases.Dictyostelium discoideum MHCK A 3 is a highly specialized protein kinase that targets three threonine residues located in the ␣-helical coiled-coil tail of myosin II (1-4). Phosphorylation of these sites results in the disassembly of myosin II bipolar filaments and inhibits processes, such as cytokinesis, that depend on myosin II contractile activity (5, 6). MHCK A is 130-kDa in size and consists of an N-terminal ␣-helical coiled-coil domain, a central kinase domain, and a C-terminal WD-repeat domain (7). The coiled-coil domain assembles into trimers or tetramers, binds, and cross-links actin filaments and is responsible for targeting MHCK A to actin-rich cellular protrusions (8 -10). The WD-repeat domain interacts with filamentous myosin II and is required for MHCK A to efficiently phosphorylate myosin II (10, 11). The kinase domain of MHCK A bears no sequence similarity to the superfamily of "conventional" eukaryotic protein kinases but instead belongs to a small but widespread family of atypical protein kinases termed the ␣-kinases (12).In addition to MHCK A, D. discoideum expresses five proteins with ␣-kinase domains. Three of the proteins, termed MHCK B, C, and D, are closely related to MHCK A and at least two to them, MHCK B and C, function cooperatively to regulate myosin II filament assembly in vivo (13-15). The other two ␣-kinases, AK1 and VwkA, have domain structures unrelated to MHCK A...

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

“…A-CAT hydrolyzed ATP with a k cat of 1.92 min Ϫ1 , which is similar to the value previously determined by following release of 32 P i from [␥ 32 P]ATP (Fig. 2 and Table 3) (36). ADP and AMP were hydrolyzed at rates 3-and 6-fold slower than ATP, respectively ( Fig.…”

“…Results represent the mean and standard deviation of at least three separate experiments. tion site (Thr-825) required for activity (28,36). Crystallization of A-CAT in the presence of MgATP has yielded structures with ADP (A-CAT⅐ADP⅐P) and AMP (A-CAT⅐AMP) bound to the inter-lobe cleft (Table 1) (19).…”

A Conserved Loop in the Catalytic Domain of Eukaryotic Elongation Factor 2 Kinase Plays a Key Role in Its Substrate Specificity

“…1C) (11). Mutating any of these residues (to alanine) almost abolished MHCK A activity (11). Thr825 corresponds to Thr348 in eEF2K.…”

“…However, in our studies, only two major sites of autophosphorylation were evident (13), suggesting that further autophosphorylation involves lowlevel phosphorylation at multiple sites. One of the major sites, a threonine C-terminal to the kinase domain (Thr348 in human eEF2K), is conserved in MHCK A and appears to play a critical role in kinase activation (11,13). Structural and other studies from Côté's group suggest that, for MHCK A, the phosphothreonine docks into a phosphate-binding pocket to induce an active conformation (11).…”

“…eEF2K and other ␣-kinases undergo autophosphorylation at multiple sites (10)(11)(12)(13)(14)(15). For eEF2K, this is an intramolecular reaction (14), and at extended times, several moles of phosphate are incorporated per mole of eEF2K protein (13,15).…”