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...