RESUMO
The Mediterranean fruit fly Ceratitis capitata is an insect capable of wreaking extensive damage to a wide range of fruit crops. Protein kinase CK2 is a ubiquitous Ser/Thr kinase that is highly conserved among eukaryotes; it is a heterotetramer composed of two catalytic (α) and a dimer of regulatory (ß) subunits. We present here the construction of the cDNA molecules of the CK2α and CK2ß subunits from the medfly C. capitata by the 5'/3' RACE and RT-PCR methods, respectively. CcCK2α catalytic subunit presents the characteristic and conserved features of a typical protein kinase, similar to the regulatory CcCK2ß subunit, that also possess the conserved features of regulatory CK2ß subunits, as revealed by comparison of their predicted amino acid sequences with other eukaryotic species. The recombinant CcCK2α and CcCK2ß proteins were purified by affinity chromatography to homogeneity, after overexpression in Escherichia coli. CcCK2α is capable to utilize GTP and its activity and is inhibited by polyanions and stimulated by polycations in phosphorylation assays, using purified acidic ribosomal protein P1 as a substrate.
Assuntos
Caseína Quinase II/genética , Caseína Quinase II/isolamento & purificação , Ceratitis capitata/enzimologia , Ceratitis capitata/genética , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Sequência de Aminoácidos , Animais , Caseína Quinase II/química , Domínio Catalítico , Cromatografia de Afinidade , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Proteínas Imobilizadas/metabolismo , Dados de Sequência Molecular , Subunidades Proteicas/química , Proteínas Recombinantes/genética , Alinhamento de SequênciaRESUMO
Protein kinase CK2 is a ubiquitous, highly pleiotropic, and constitutively active phosphotransferase that phosphorylates mainly serine and threonine residues. CK2 has been studied and characterized in many organisms, from yeast to mammals. The holoenzyme is generally composed of two catalytic (α and/or α') and two regulatory (ß) subunits, forming a differently assembled tetramer. The free and catalytically active α/α' subunits can be present in cells under some circumstances. We present here the isolation of a putative catalytic CK2α subunit and holoenzyme from gills of the mussel Mytilus galloprovincialis capable of phosphorylating the purified recombinant ribosomal protein rMgP1. For further analysis of M. galloprovincialis protein kinase CK2, the cDNA molecules of CK2α and CK2ß subunits were constructed and cloned into expression vectors, and the recombinant proteins were purified after expression in Escherichia coli. The recombinant MgCK2ß subunit and MgP1 were phosphorylated by the purified recombinant MgCK2α subunit. The mussel enzyme presented features typical for CK2: affinity for GTP, inhibition by both heparin and ATP competitive inhibitors (TBBt, TBBz), and sensitivity towards NaCl. Predicted amino acid sequence comparison showed that the M. galloprovincialis MgCK2α and MgCK2ß subunits have similar features to their mammalian orthologs.
Assuntos
Caseína Quinase II/genética , Caseína Quinase II/isolamento & purificação , DNA Complementar/biossíntese , Mytilus/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , Brânquias/enzimologia , Dados de Sequência Molecular , Fosfoproteínas/metabolismo , Fosforilação , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Proteínas Ribossômicas/metabolismo , Análise de Sequência de DNA , Homologia de SequênciaRESUMO
The stalk, a characteristic structure of the large ribosomal subunit, is directly involved in the interaction with the soluble factors during translation. In the Mediterranean mussel Mytilus galloprovincialis, the stalk consists of one 32 kDa protein, MgP0, and two smaller, 12 kDa acidic proteins, MgP1 and MgP2, of pI 3.0 and 4.0, respectively, as revealed by analysis of purified ribosomes with electrophoresis and Western blot with a specific monoclonal antibody. Treatment of the ribosomes with alkaline phosphatase showed movement of the bands corresponding to the acidic MgP1 and MgP2 proteins to more basic pH after isoelectrofocusing, implying phosphorylation. The cDNA molecules of M. galloprovincialis ribosomal proteins MgP0, MgP1 and MgP2 and superoxide dismutase (MgSOD) were isolated from a cDNA library or constructed by RT-PCR, cloned in expression vectors and expressed in Escherichia coli. The recombinant proteins were purified with immobilized metal ion affinity chromatography (IMAC) and identified with immunoblotting. Exposure of mussels at cadmium and sorbitol and analysis of gill tissue extracts showed over expression of MgP0 protein.
Assuntos
Mytilus/enzimologia , Mytilus/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Estresse Fisiológico , Superóxido Dismutase/genética , Sequência de Aminoácidos , Animais , Cádmio/farmacologia , Cromatografia de Afinidade , Clonagem Molecular , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Immunoblotting , Dados de Sequência Molecular , Mytilus/efeitos dos fármacos , Proteínas Ribossômicas/química , Proteínas Ribossômicas/isolamento & purificação , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sorbitol/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Superóxido Dismutase/química , Superóxido Dismutase/isolamento & purificaçãoRESUMO
In the silkworm Bombyx mori the ribosomal stalk P-protein family consists of two low MW acidic proteins, BmP1 and BmP2, and of one higher MW protein, BmP0, as shown by electrophoretical and immunoblotting western blot analysis of purified ribosomes. Treatment of ribosomes with alkaline phosphatase followed by electrofocusing shifted the isoelectric points to higher pH, implying phosphorylation of the proteins. The cDNAs encoding BmP1 and BmP2 proteins were constructed and expressed in the Saccharomyces cerevisiae mutant strains defective in either the endogenous P1 or P2 proteins. The recombinant silkworm proteins could complement the absence of the homologous yeast proteins and were incorporated to the ribosomes of the transformed strains, helping the binding of the remaining endogenous acidic proteins, present in the cytoplasm in different extent. Thus, BmP1 was able to replace YP1alpha, preferentially binding YP2beta to the ribosome, while BmP2 replaced both yeast P2 proteins and induced the binding of both YP1alpha and YP1beta.
