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3.
J Med Chem ; 48(16): 5092-5, 2005 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-16078826
4.
Biochemistry ; 44(28): 9563-73, 2005 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-16008341

RESUMO

Protein kinase C theta (PKCtheta), a member of the Ca(2+)-independent novel subfamily of PKCs, is required for T-cell receptor (TCR) signaling and IL2 production. PKCtheta-deficient mice have impaired Th2 responses in a murine ova-induced asthma model, while Th1 responses are normal. As an essential component of the TCR signaling complex, PKCtheta is a unique T-cell therapeutic target in the specific treatment of T-cell-mediated diseases. We report here the PKCtheta autophosphorylation characteristics and elucidation of the catalytic mechanism of the PKCtheta kinase domain using steady-state kinetics. Key phosphorylated residues of the active PKCtheta kinase domain expressed in Escherichia coli were characterized, and mutational analysis of the kinase domain was performed to establish the autophosphorylation and kinase activity relationships. Initial velocity, product inhibition, and dead-end inhibition studies provided assignments of the kinetic mechanism of PCKtheta(362)(-)(706) as ordered, wherein ATP binds kinase first and ADP is released last. Effects of solvent viscosity and ATPgammaS on PKCtheta catalysis demonstrated product release is partially rate limiting. Our studies provide important mechanistic insights into kinase activity and phosphorylation-mediated regulation of the novel PKC isoform, PKCtheta. These results should aid the design and discovery of PKCtheta antagonists as therapeutics for modulating T-cell-mediated immune and respiratory diseases.


Assuntos
Domínio Catalítico , Isoenzimas/química , Isoenzimas/metabolismo , Proteína Quinase C/química , Proteína Quinase C/metabolismo , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Ligação Competitiva , Catálise , Domínio Catalítico/genética , Ativação Enzimática , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Concentração Osmolar , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteína Quinase C-theta , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/química , Serina/genética , Especificidade por Substrato , Treonina/química , Treonina/genética , Treonina/metabolismo
6.
J Am Chem Soc ; 126(46): 15106-19, 2004 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-15548008

RESUMO

We present the structure-based optimization of a series of estrogen receptor-beta (ERbeta) selective ligands. X-ray cocrystal structures of these ligands complexed to both ERalpha and ERbeta are described. We also discuss how molecular modeling was used to take advantage of subtle differences between the two binding cavities in order to optimize selectivity for ERbeta over ERalpha. Quantum chemical calculations are utilized to gain insight into the mechanism of selectivity enhancement. Despite only two relatively conservative residue substitutions in the ligand binding pocket, the most selective compounds have greater than 100-fold selectivity for ERbeta relative to ERalpha when measured using a competitive radioligand binding assay.


Assuntos
Receptor beta de Estrogênio/química , Receptor beta de Estrogênio/metabolismo , Sequência de Aminoácidos , Benzofuranos/química , Benzofuranos/metabolismo , Benzoxazóis/química , Benzoxazóis/metabolismo , Sítios de Ligação , Ligação Competitiva , Cristalografia por Raios X , Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/metabolismo , Humanos , Ligantes , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Teoria Quântica , Ensaio Radioligante , Relação Estrutura-Atividade , Especificidade por Substrato
7.
J Biol Chem ; 279(48): 50401-9, 2004 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-15364937

RESUMO

A member of the novel protein kinase C (PKC) subfamily, PKC, is an essential component of the T cell synapse and is required for optimal T cell activation and interleukin-2 production. Selective involvement of PKC in TCR signaling makes this enzyme an attractive therapeutic target in T cell-mediated disease processes. In this report we describe the crystal structure of the catalytic domain of PKC at 2.0-A resolution. Human recombinant PKC kinase domain was expressed in bacteria as catalytically active phosphorylated enzyme and co-crystallized with its subnanomolar, ATP site inhibitor staurosporine. The structure follows the classic bilobal kinase fold and shows the enzyme in its active conformation and phosphorylated state. Inhibitory interactions between conserved features of staurosporine and the ATP-binding cleft are accompanied by closing of the glycine-rich loop, which also maintains an inhibitory arrangement by blocking the phosphate recognition subsite. The two major phosphorylation sites, Thr-538 in the activation loop and Ser-695 in the hydrophobic motif, are both occupied in the structure, playing key roles in stabilizing active conformation of the enzyme and indicative of PKC autocatalytic phosphorylation and activation during bacterial expression. The PKC-staurosporine complex represents the first kinase domain crystal structure of any PKC isotypes to be determined and as such should provide valuable insight into PKC specificity and into rational drug design strategies for PKC selective leads.


Assuntos
Isoenzimas/química , Proteína Quinase C/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Cinética , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Proteína Quinase C-theta , Estrutura Terciária de Proteína , Alinhamento de Sequência , Estaurosporina/metabolismo , Especificidade por Substrato
9.
Structure ; 11(6): 627-36, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12791252

RESUMO

MAP KAP kinase 2 (MK2), a Ser/Thr kinase, plays a crucial role in the inflammatory process. We have determined the crystal structures of a catalytically active C-terminal deletion form of human MK2, residues 41-364, in complex with staurosporine at 2.7 A and with ADP at 3.2 A, revealing overall structural similarity with other Ser/Thr kinases. Kinetic analysis reveals that the K(m) for ATP is very similar for MK2 41-364 and p38-activated MK2 41-400. Conversely, the catalytic rate and binding for peptide substrate are dramatically reduced in MK2 41-364. However, phosphorylation of MK2 41-364 by p38 restores the V(max) and K(m) for peptide substrate to values comparable to those seen in p38-activated MK2 41-400, suggesting a mechanism for regulation of enzyme activity.


Assuntos
Difosfato de Adenosina/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Estaurosporina/metabolismo , Sequência de Aminoácidos , Ativação Enzimática , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Substâncias Macromoleculares , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Alinhamento de Sequência , Proteínas Quinases p38 Ativadas por Mitógeno
10.
J Virol ; 76(10): 5156-66, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-11967331

RESUMO

Simian virus 40 (SV40) enters cells by atypical endocytosis mediated by caveolae that transports the virus to the endoplasmic reticulum (ER) instead of to the endosomal-lysosomal compartment, which is the usual destination for viruses and other cargo that enter by endocytosis. We show here that SV4O is transported to the ER via an intermediate compartment that contains beta-COP, which is best known as a component of the COPI coatamer complexes that are required for the retrograde retrieval pathway from the Golgi to the ER. Additionally, transport of SV40 to the ER, as well as infection, is sensitive to brefeldin A. This drug acts by specifically inhibiting the ARF1 GTPase, which is known to regulate assembly of COPI coat complexes on Golgi cisternae. Moreover, some beta-COP colocalizes with intracellular caveolin-1, which was previously shown to be present on a new organelle (termed the caveosome) that is an intermediate in the transport of SV40 to the ER (L. Pelkmans, J. Kartenbeck, and A. Helenius, Nat. Cell Biol. 3:473-483, 2001). We also show that the internal SV40 capsid proteins VP2 and VP3 become accessible to immunostaining starting at about 5 h. Most of that immunostaining overlays the ER, with some appearing outside of the ER. In contrast, immunostaining with anti-SV40 antisera remains confined to the ER.


Assuntos
Antivirais/farmacologia , Brefeldina A/farmacologia , Cavéolas/virologia , Retículo Endoplasmático/virologia , Vírus 40 dos Símios/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Capsídeo/análise , Capsídeo/metabolismo , Cavéolas/metabolismo , Proteína Coatomer/análise , Proteína Coatomer/metabolismo , Endocitose , Retículo Endoplasmático/metabolismo , Replicação Viral
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