RESUMO
Dictyostelium discoideum MyoB is a single-headed class I myosin. Analysis of purified MyoB by SDS-PAGE indicated the presence of an approximately 9-kDa light chain. A tryptic digest of MyoB yielded a partial sequence for the light chain that exactly matched a sequence in a 73-amino acid, 8,296-Da protein (dictyBase number DDB0188713). This protein, termed MlcB, contains two EF-hand motifs and shares approximately 30% sequence identity with the N- and C-terminal lobes of calmodulin. FLAG-MlcB expressed in Dictyostelium co-immunoprecipitated with MyoB but not with the related class myosins and MyoD. Recombinant MlcB bound Ca2+ with a Kd value of 0.2 microm and underwent a Ca2+-induced change in conformation that increased alpha-helical content and surface hydrophobicity. Mutational analysis showed that the first EF-hand was responsible for Ca2+ binding. In the presence and absence of Ca2+ MlcB was a monomer in solution and bound to a MyoB IQ motif peptide with a Kd value of approximately 0.5 microm. A MyoB head-neck construct with a Ser to Glu mutation at the TEDS site bound MlcB and displayed an actin-activated Mg2+ ATPase activity that was insensitive to Ca2+. We conclude that MlcB represents a novel type of small myosin light chain that binds to IQ motifs in a manner comparable with a single lobe of a typical four-EF-hand protein.
Assuntos
Dictyostelium , Cadeias Leves de Miosina/química , Miosina Tipo I/química , Proteínas de Protozoários/química , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Cromatografia Líquida de Alta Pressão , Motivos EF Hand , Eletroforese em Gel de Poliacrilamida , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Peso Molecular , Cadeias Leves de Miosina/classificação , Cadeias Leves de Miosina/genética , Miosina Tipo I/genética , Oligopeptídeos , Peptídeos , Ligação Proteica , Conformação Proteica , Proteínas de Protozoários/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Dictyostelium discoideum MyoD, a long-tailed class I myosin, co-purified with two copies of a 16 kDa light chain. Sequence analysis of the MyoD light chain showed it to be a unique protein, termed MlcD, that shares 44% sequence identity with Dictyostelium calmodulin and 43% sequence identity with Acanthamoeba castellanii myosin IC light chain. MlcD comprises four EF-hands; however, EF-hands 2-4 contain mutations in key Ca2+-co-ordinating residues that would be predicted to impair Ca2+ binding. Electrospray ionization MS of MlcD in the presence of Ca2+ and La3+ showed the presence of one major and one minor metal-binding site. MlcD contains a single tryptophan residue (Trp39), the fluorescence intensity of which was quenched upon addition of Ca2+ or Mg2+, yielding apparent dissociation constants ( K'(d)) of 52 microM for Ca2+ and 450 microM for Mg2+. The low affinity of MlcD for Ca2+ indicates that it cannot function as a sensor of physiological Ca2+. Ca2+ did not affect the binding of MlcD to MyoD or to either of the two MyoD IQ (Ile-Gln) motifs. FLAG-MlcD expressed in Dictyostelium formed a complex with MyoD, but not with the two other long-tailed Dictyostelium myosin I isoenzymes, MyoB and MyoC. Through its specific association with the Ca2+-insensitive MlcD, MyoD may exhibit distinct regulatory properties that distinguish it from myosin I isoenzymes with calmodulin light chains.
Assuntos
Proteínas de Ligação ao Cálcio/química , Dictyostelium/química , Proteína MyoD/química , Cadeias Leves de Miosina/química , Miosina Tipo I/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Calmodulina/metabolismo , Proteínas de Ligação a Calmodulina/química , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Dictyostelium/genética , Dados de Sequência Molecular , Proteína MyoD/genética , Proteína MyoD/metabolismo , Cadeias Leves de Miosina/genética , Cadeias Leves de Miosina/metabolismo , Miosina Tipo I/biossíntese , Miosina Tipo I/genética , Miosina Tipo I/metabolismo , Filogenia , Proteínas de Protozoários/química , Proteínas de Protozoários/genéticaRESUMO
Diacylglycerol kinases (DGKs) phosphorylate the neutral lipid diacylglycerol (DG) to produce phosphatidic acid (PA). In mammalian systems DGKs are a complex family of at least nine isoforms that are thought to participate in down-regulation of DG-based signalling pathways and perhaps activation of PA-stimulated signalling events. We report here that the simple protozoan amoeba Dictyostelium discoideum appears to contain a single gene encoding a DGK enzyme. This gene, dgkA, encodes a deduced protein that contains three C1-type cysteine-rich repeats, a DGK catalytic domain most closely related to the theta subtype of mammalian DGKs and a C-terminal segment containing a proline/glutamine-rich region and a large aspargine-repeat region. This gene corresponds to a previously reported myosin II heavy chain kinase designated myosin heavy chain-protein kinase C (MHC-PKC), but our analysis clearly demonstrates that this protein does not, as suggested by earlier data, contain a protein kinase catalytic domain. A FLAG-tagged version of DgkA expressed in Dictyostelium displayed robust DGK activity. Earlier studies indicating that disruption of this locus alters myosin II assembly levels in Dictyostelium raise the intriguing possibility that DG and/or PA metabolism may play a role in controlling myosin II assembly in this system.
Assuntos
Diacilglicerol Quinase/química , Diacilglicerol Quinase/genética , Algoritmos , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Domínio Catalítico , Clonagem Molecular , Cisteína/química , DNA Complementar/metabolismo , Dictyostelium , Regulação para Baixo , Humanos , Modelos Genéticos , Dados de Sequência Molecular , Miosina Tipo II/química , Miosina Tipo II/metabolismo , Ácidos Fosfatídicos/metabolismo , Filogenia , Reação em Cadeia da Polimerase , Testes de Precipitina , Ligação Proteica , Isoformas de Proteínas , Proteína Quinase C/metabolismo , Estrutura Terciária de Proteína , RNA/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
Dictyostelium myosin II is a conventional, two-headed myosin that consists of two copies each of a myosin heavy chain (MHC), an essential light chain (ELC) and a regulatory light chain (RLC). The MHC is comprised of an amino-terminal motor domain, a neck region that binds the RLC and ELC and a carboxyl-terminal alpha-helical coiled-coil tail. Electrostatic interactions between the tail domains mediate the self-assembly of myosin II into bipolar filaments that are capable of interacting with actin filaments to generate a contractile force. In this review we discuss the regulation of Dictyostelium myosin II by a myosin light chain kinase (MLCK-A) that phosphorylates the RLC and increases motor activity and by MHC kinases (MHCKs) that phosphorylate the tail and prevent filament assembly. Dictyostelium may express as many as four MHCKs (MHCK A-D) consisting of an atypical alpha-kinase catalytic domain and a carboxyl-terminal WD repeat domain that targets myosin II filaments. A previously reported MHCK, termed MHC-PKC, now seems more likely to be a diacylglycerol kinase (DgkA). The relationship of the MHCKs to the larger family of alpha-kinases is discussed and key features of the structure of the alpha-kinase catalytic domain are reviewed. Potential upstream regulators of myosin II are described, including DgkA, cGMP, cAMP and PAKa, a target for Rac GTPases. Recent results point to a complex network of signaling pathways responsible for controling the activity and localization of myosin II in the cell.