Identification and Characterization of an 8-kDa Light Chain Associated with Dictyostelium discoideum MyoB, a Class I Myosin

Crawley, Scott W.; Lee, Sheu-Fen; Li, Zhihao; Chitayat, Seth; Smith, Steven P.; Côté, Graham P.

doi:10.1074/jbc.m508670200

Cited by 18 publications

(48 citation statements)

References 48 publications

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“…Cells were lysed by homogenization in 500 mM NaCl, 1 mM EDTA, 0.3% Triton X-100, and 50 mM Tris-HCl, pH 8.0, containing one complete mini protease inhibitor tablet (Roche Applied Diagnostics) per 50 ml of buffer at 4°C. The supernatant obtained following centrifugation at 12,000 ϫ g for 1 h was passed over a column of anti-FLAG M2 Affinity gel (Sigma) (33). FLAG-MHCK A was eluted with TBS buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4) containing 200 g/ml of FLAG peptide and dialyzed against 20 mM NaCl and 50 mM Tris-HCl, pH 7.4.…”

Section: Methodsmentioning

confidence: 99%

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

Crawley

Gharaei

et al. 2011

Journal of Biological Chemistry

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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...

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Section: Methodsmentioning

confidence: 99%

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

Crawley

Gharaei

et al. 2011

Journal of Biological Chemistry

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“…Given that most of the class I myosins studied to date employ one or more calmodulins (Kim and Flavell, 2008), it is not clear why Myo1p utilizes Cam2p. Nevertheless, calmodulin-related light chains appear to be physiologically significant because they have been found to associate with certain other class I myosins: Acanthamoeba myosin-IC associates with alternative light chain MICLC (Wang et al, 1997); Dictyostelium MyoB and MyoD associate with MlcB and MlcD, respectively (Crawley et al, 2006;De La Roche et al, 2003); whereas vertebrate myosin-1c can interact with two other light chains (CIB1 and CaBP1) in addition to calmodulin (Tang et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

A calmodulin-related light chain from fission yeast that functions with myosin-I and PI 4-kinase

Sammons

James

Clayton

et al. 2011

Journal of Cell Science

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SummaryFission yeast myosin-I (Myo1p) not only associates with calmodulin, but also employs a second light chain called Cam2p. cam2 cells exhibit defects in cell polarity and growth consistent with a loss of Myo1p function. Loss of Cam2p leads to a reduction in Myo1p levels at endocytic patches and a 50% drop in the rates of Myo1p-driven actin filament motility. Thus, Cam2p plays a significant role in Myo1p function. However, further studies indicated the existence of an additional Cam2p-binding partner. Cam2p was still present at cortical patches in myo1 cells (or in myo1-IQ2 mutants, which lack an intact Cam2p-binding motif), whereas a cam2 null (cam2) suppressed cytokinesis defects of an essential light chain (ELC) mutant known to be impaired in binding to PI 4-kinase (Pik1p). Binding studies revealed that Cam2p and the ELC compete for Pik1p. Cortical localization of Cam2p in the myo1 background relied on its association with Pik1p, whereas overexpression studies indicated that Cam2p, in turn, contributes to Pik1p function. The fact that the Myo1p-associated defects of a cam2 mutant are more potent than those of a myo1-IQ2 mutant suggests that myosin light chains can contribute to actomyosin function both directly and indirectly (via phospholipid synthesis at sites of polarized growth).

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“…The LCBDs of myosin-1B and myosin-1C contain one and three IQ motifs, respectively, with the IQ motif of the former displaying specific binding to the small (i.e. ϳ8.5 kDa) LC MlcB (25). The first two IQ motifs of myosin-1C are divergent from the consensus IQ sequences in that they are 18 amino acid residues in length, and the conserved glutamine is substituted for a lysine and is specifically recognized by the LC MlcC (23).…”

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confidence: 99%

“…The first two IQ motifs of myosin-1C are divergent from the consensus IQ sequences in that they are 18 amino acid residues in length, and the conserved glutamine is substituted for a lysine and is specifically recognized by the LC MlcC (23). The identity of the LC that binds IQ3 of myosin-1C is not currently known.MlcB and MlcC consist of two helix-loop-helix EF-hand motifs connected by a short linker sequence, and particularly relevant to the biological function are monomers in solution (23,25,26 …”

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Structure of the Single-lobe Myosin Light Chain C in Complex with the Light Chain-binding Domains of Myosin-1C Provides Insights into Divergent IQ Motif Recognition

Langelaan¹,

Liburd²,

Yang

et al. 2016

Journal of Biological Chemistry

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Myosin light chains are key regulators of class 1 myosins and typically comprise two domains, with calmodulin being the archetypal example. They bind IQ motifs within the myosin neck region and amplify conformational changes in the motor domain. A single lobe light chain, myosin light chain C (MlcC), was recently identified and shown to specifically bind to two sequentially divergent IQ motifs of the Dictyostelium myosin-1C. To provide a molecular basis of this interaction, the structures of apo-MlcC and a 2:1 MlcC⅐myosin-1C neck complex were determined. The two non-functional EF-hand motifs of MlcC pack together to form a globular four-helix bundle that opens up to expose a central hydrophobic groove, which interacts with the N-terminal portion of the divergent IQ1 and IQ2 motifs. The N-and C-terminal regions of MlcC make critical contacts that contribute to its specific interactions with the myosin-1C divergent IQ motifs, which are contacts that deviate from the traditional mode of calmodulin-IQ recognition.The class 1 myosins (myosin-1) are a widely expressed family of single-headed, non-filament-forming, membrane-binding myosins (1-3). The myosin-1 heavy chain consists of a highly conserved N-terminal motor domain with sites for binding actin and for ATP hydrolysis, a light chain-binding domain (LCBD) 5 that acts as the motor's lever arm, and a C-terminal tail. The myosin-1 tail contains a tail homology 1 (TH1) domain that interacts with acidic phospholipids and, in some cases, also contains a glycine-and proline-rich TH2 domain that binds filamentous actin and an Src homology 3 domain that forms complexes with proteins that stimulate actin filament assembly (4 -10). Myosin-1 is able to cross-link the plasma membrane to the underlying actin cytoskeleton and plays a direct role in the control of cortical and membrane tension (11,12). These motor proteins can also drive vesicle trafficking, pseudopod retraction, and the uptake of particles and fluids by phagocytosis and micropinocytosis (13-16).The myosin-1 LCBD includes one or more IQ motifs, which are ␣-helical sequences between 18 and 25 residues in length that terminate with a loosely conserved IQXXXRGXXXR consensus sequence (17, 18). IQ motifs bind calmodulin (CaM) or CaM-related light chains (LCs) in the absence of Ca 2ϩ . The LCs stabilize the ␣-helical structure of the LCBD, allowing it to function as a rigid swinging lever arm to amplify ATP-dependent conformational changes that originate within the motor domain (19). The LCs are also important regulatory sites that interact with the motor domain to influence mechanochemical properties such as step size, motility rate, and force sensing (1,20,21).The social amoeba Dictyostelium discoideum has been used extensively to study myosin-1 structure, function, and regulation. The seven Dictyostelium myosin-1 isozymes include three with short tails that contain only a TH1 domain (myosin-1A, -1E, and -1F), three with long tails that have TH1, TH2, and Src homology 3 domains (myosin-1B, -1C, and -1D), and one ...

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Identification and Characterization of an 8-kDa Light Chain Associated with Dictyostelium discoideum MyoB, a Class I Myosin

Abstract: Dictyostelium discoideum

Cited by 18 publications

References 48 publications

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

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

A calmodulin-related light chain from fission yeast that functions with myosin-I and PI 4-kinase

Structure of the Single-lobe Myosin Light Chain C in Complex with the Light Chain-binding Domains of Myosin-1C Provides Insights into Divergent IQ Motif Recognition

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