RESUMEN
Calmodulin (CaM) is an essential protein that serves as a ubiquitous intracellular receptor for Ca(2+). The Ca(2+)/CaM complex initiates a plethora of signaling cascades that culminate in alteration of cellular functions. Among the many Ca(2+)/CaM-binding proteins to be discovered, the multifunctional protein kinases CaMKI, II, and IV play pivotal roles. Our review focuses on this class of CaM kinases to illustrate the structural and biochemical basis for Ca(2+)/CaM interaction with and regulation of its target enzymes. Gene transcription has been chosen as the functional endpoint to illustrate the recent advances in Ca(2+)/CaM-mediated signal transduction mechanisms.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Secuencia de Aminoácidos , Animales , Señalización del Calcio/fisiología , Activación Enzimática/fisiología , Humanos , Datos de Secuencia MolecularRESUMEN
Using recently refined diagnostic criteria, the authors hypothesized that the frequent touching of others by a 16-year-old male adolescent with Tourette's syndrome was a compulsion and not a tic. Consistent with the study's hypothesis, the authors applied exposure and response prevention, a procedure empirically supported for treatment of compulsions but not for tics, to the touching. Results showed a significant decrease in touching attempts, overt anxiety, and subjective anxiety across time.
Asunto(s)
Terapia Conductista/métodos , Conducta Compulsiva/terapia , Trastorno Obsesivo Compulsivo/complicaciones , Trastorno Obsesivo Compulsivo/terapia , Síndrome de Tourette/complicaciones , Adolescente , Humanos , Masculino , Síndrome de Tourette/terapia , Resultado del TratamientoRESUMEN
Transforming growth factor-beta (TGFbeta) enhances transcription from reporter genes regulated by a single consensus cAMP-response element (CRE) upon transfection into the immortalized human keratinocyte cell line, HaCaT. Whereas both CRE-binding protein (CREB) and c-Jun present in extracts of unstimulated cells can complex with a CRE in gel-shift experiments, TGFbeta treatment increases the amount of c-Jun found in the complex. Overexpression of c-Jun is sufficient to increase CRE and GAL4-CREB-dependent transcription and mimics the stimulatory effects of TGFbeta on transcription from either reporter gene. Surprisingly, although a portion of CREB in unstimulated cells is phosphorylated on the activating serine residue, Ser-133, this level of phospho-CREB is not altered by TGFbeta treatment. In fact, the CREB-dependent transcriptional effects of TGFbeta or c-Jun do not require phosphorylation of Ser-133, although CREB-binding protein (CBP) is required as evidenced by the observation that the adenoviral oncoprotein E1A can block the effects of both agents. c-Jun enhancement of CRE or GAL4-CREB-dependent transcription neither requires the DNA-binding nor N-terminal domains of c-Jun. Collectively, these results are consistent with a model in which signaling pathways initiated by TGFbeta can stimulate CREB-dependent transcription by increasing the cellular concentration of c-Jun, which participates in activation of the CBP-containing transcription complex.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , ADN/metabolismo , Proteínas Proto-Oncogénicas c-jun/fisiología , Transcripción Genética , Factor de Crecimiento Transformador beta/farmacología , Proteínas E1A de Adenovirus/farmacología , Línea Celular Transformada , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/farmacología , Sinergismo Farmacológico , Expresión Génica , Humanos , Queratinocitos/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/farmacología , TransfecciónRESUMEN
Phosphorylation of Ca2+/calmodulin-dependent protein kinase I (CaM KI) at Thr-177 by recombinant rat Ca2+/calmodulin-dependent kinase kinase B (CaM KKB) modulates the kinetics of synapsin-(4-13) peptide phosphorylation by reducing the Km 44-fold and decreasing the KCaM 4-fold. There is also a slight decrease in Km for ATP and increase in enzyme Vmax. A synthetic peptide substrate from the yeast transcription factor, ADR1-(222-234)G233 is a 15-fold better substrate for the Thr-177 dephospho-form of CaM KI than synapsin-(4-13). The Thr-177 dephospho-enzyme has a Km and Vmax for ADR1-(222-234)G233 similar to the values with synapsin-(4-13) using the Thr-177 phosphorylated enzyme. Likewise, with ADR1-(222-234)G233 as substrate, phosphorylation of Thr-177 or substitution of T177A had very little effect on the kinetic values. Using chimeric peptides between synapsin-(4-13) and ADR1-(222-234)G233 we found that N-terminal basic residues at P-7 and P-6 positions were sufficient to allow efficient phosphorylation by the Thr-177 dephospho-form of CaM KI. Phosphorylation of Thr-177 expands the substrate specificity of CaM KI and is not merely an "on-off" switch for kinase activity.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Catálisis , Activación Enzimática , Células HL-60 , Humanos , Cinética , Fosforilación , Ratas , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) is a monomeric multifunctional enzyme that is expressed only in subanatomical portions of the brain, T lymphocytes, and postmeiotic male germ cells. It is present in the nucleus of the cells in which it is expressed and can phosphorylate and activate the cyclic AMP response element binding proteins CREB and CREM tau in a manner analogous to protein kinase A. In the absence of Ca2+/calmodulin, CaMKIV is inactive. Activation requires three events: 1) binding of Ca2+/calmodulin; 2) phosphorylation of a single threonine residue present in the activation loop by a separate protein kinase that is also Ca2+/calmodulin-dependent; and 3) autophosphorylation of serine residues present in the extreme N-terminus that is required to relieve a novel form of autoinhibition. The gene for rat CaMKIV has been cloned and found to span 42 kb of DNA. The gene encodes three proteins: namely, the alpha and beta forms of CaMKIV that differ only in that the beta form contains a 28 amino acid N-terminal extension as well as calspermin. Calspermin is the C-terminal 169 amino acids of CaMKIV that binds Ca2+/calmodulin and is expressed only in postmeiotic male germ cells. The promoter for calspermin resides in the penultimate intron of the CaMKIV gene and is regulated by two CREs. This promoter is sufficient to faithfully target expression of a reporter gene to the postmeiotic male germ cells of transgenic mice. Transgene expression can be induced in cells from the transgenic mice that do not normally express it by transfection of CREM tau and CaMKIV. These data suggest that rearrangement of chromatin during meiosis together with the expression of CREM tau at high levels are sufficient to control expression of the calspermin promoter during spermatogenesis. On the other hand, the developmental expression of CaMKIV in brain and thymus appears to be controlled by thyroid hormone mediated via the thyroid hormone receptor alpha. In T lymphocytes, CaMKIV will phosphorylate CREB in response to signals that result in T cell activation. Transgenic mice that express a kinase minus mutant of CaMKIV specifically in thymic T cells show a marked reduction of total thymic cellularity. The remaining T cells undergo a much greater than normal rate of spontaneous apoptosis when placed in culture. These cells fail to generate the signals to phosphorylate CREB and produce significantly less of the cytokine Interleukin-2 (IL-2) in response to agents that either increase intracellular Ca2+ and/or activate protein kinase C. Collectively, the data suggest that CaMKIV may be involved both in preventing apoptosis during T cell development and also in the early cascade of events that is required to activate the mature T cells in response to a mitogenic stimulus.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Regulación Enzimológica de la Expresión Génica , Secuencia de Aminoácidos , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Proteínas de Unión a Calmodulina/genética , Proteínas de Unión a Calmodulina/fisiología , Activación de Linfocitos , Masculino , Ratones , Ratones Transgénicos , Ratas , Linfocitos T/fisiologíaRESUMEN
We have purified to near homogeneity from rat brain two Ca(2+)-calmodulin-dependent protein kinase I (CaM kinase I) activating kinases, termed here CaM kinase I kinase-alpha and CaM kinase I kinase-beta (CaMKIK alpha and CaMKIK beta, respectively). Both CaMKIK alpha and CaMKIK beta are also capable of activating CaM kinase IV. Activation of CaM kinase I and CaM kinase IV occurs via phosphorylation of an equivalent Thr residue within the "activation loop" region of both kinases, Thr-177 and Thr-196, respectively. The activities of CaMKIK alpha and CaMKIK beta are themselves strongly stimulated by the presence of Ca(2+)-CaM, and both appear to be capable of Ca(2+)-CaM-dependent autophosphorylation. Automated microsequence analysis of the purified enzymes established that CaMKIK alpha and -beta are the products of distinct genes. In addition to rat, homologous nucleic acids corresponding to these CaM kinase kinases are present in humans and the nematode, Caenorhabditis elegans. CaMKIK alpha and CaMKIK beta are thus representatives of a family of enzymes, which may function as key intermediaries in Ca(2+)-CaM-driven signal transduction cascades in a wide variety of eukaryotic organisms.
Asunto(s)
Encéfalo/enzimología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Calcio/metabolismo , Calmodulina/metabolismo , Isoenzimas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Proteínas Quinasas Dependientes de Calcio-Calmodulina/aislamiento & purificación , Cromatografía Liquida , Activación Enzimática , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Datos de Secuencia Molecular , Ratas , Ratas Sprague-Dawley , Homología de Secuencia de AminoácidoRESUMEN
Human Ca(2+)-calmodulin (CaM) dependent protein kinase I (CaMKI) encodes a 370 amino acid protein with a calculated M(r) of 41,337. The 1.5 kb CaMKI mRNA is expressed in many different human tissues and is the product of a single gene located on human chromosome 3. CaMKI 1-306, was unable to bind Ca(2+)-CaM and was completely inactive thereby defining an essential component of the CaM-binding domain to residues C-terminal to 306. CaMKI 1-294 did not bind CaM but was fully active in the absence of Ca(2+)-CaM, indicating that residues 295-306 are sufficient to maintain CaMKI in an auto-inhibited state. CaMKI was phosphorylated on Thr177 and its activity enhanced approximately 25-fold by CaMKI kinase in a Ca(2+)-CaM dependent manner. Replacement of Thr177 with Ala or Asp prevented both phosphorylation and activation by CaMKI kinase and the latter replacement also led to partial activation in the absence of CaMKI kinase. Whereas CaMKI 1-306 was unresponsive to CaMKI kinase, the 1-294 mutant was phosphorylated and activated by CaMKI kinase in both the presence and absence of Ca(2+)-CaM although at a faster rate in its presence. These results indicate that the auto-inhibitory domain in CaMKI gates, in a Ca(2+)-CaM dependent fashion, accessibility of both substrates to the substrate binding cleft and CaMKI kinase to Thr177. Additionally, CaMKI kinase responds directly to Ca(2+)-CaM with increased activity.