RESUMO
Annexins are a superfamily of calcium-dependent membrane-associated proteins which interact with phospholipids. The primary structure of Annexins I, III, VII, VIII and XI contain a region enriched in proline, glutamate, serine and threonine (PEST sequences) towards the N-terminal end while annexins II, V and VI possess PEST regions somewhat distal to the N-terminus. These PEST sequences are believed to be the signals for rapid intracellular degradation. Annexin I is known to be cleaved by calpain near its PEST region suggesting that its PEST region might be a possible calpain recognition site. Western blot analysis of annexins V and XI in rat lung homogenates suggest that these proteins are resistant to proteolysis by calpain. Annexin V was found to be stable to intrinsic lung proteases in the presence of either Ca2+ or EGTA while annexin XI was found to be partially degraded by intrinsic lung proteases in the presence of EGTA. Eight of the 10 known mammalian annexins also contain a pentapeptide sequence that is biochemically related to the KFERQ motif which is a known signal that targets protein for lysosomal proteolysis. Our data suggest that the annexins may be regulated by limited proteolysis, most likely at their N-terminal end, while most, if not all, of them might be degraded by the lysosomal pathway.
Assuntos
Anexinas/química , Anexinas/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Animais , Western Blotting , Cálcio/metabolismo , Calpaína/metabolismo , Humanos , Pulmão/metabolismo , Masculino , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosfolipídeos/metabolismo , Ratos , Ratos Sprague-Dawley , Homologia de Sequência de AminoácidosRESUMO
Calmodulin-dependent protein kinase I (CaM kinase I) is a member of the expanding class of protein kinases that are regulated by calmodulin (CaM). Its putative CaM -binding region is believed to occur within a 22-residue sequence (amino acids 299-320). This sequence was chemically synthesized and utilized for CaM interaction studies. GEl band shift assays and densitometry experiments with intact CAM kinase I and the CAM-binding domain peptide (CaMKIp) reveal that they bind in an analogous manner, giving rise to 1:1 complexes. Fluorescence analysis using dansyl-CaM showed that confirmational changes CaM on binding CaM kinase I orCaMKIp were nearly identical, suggesting that the peptide mimicked the CaM-binding ability of the intact protein. In the presence of Ca, the peptide displays an enhancement of its unique Trp fluorescence as well as a marked blue shift of the emission maximum, reflecting a transfer to a more rigid, less polar environment. Quenching studies, using acrylamide, confirmed that the Trp in the peptide on binding CaM is no longer freely exposed to solvent as is the case for the free peptide. Studies with a series of MET small mutants of CaM showed that the Trp-containing N-terminal lobe of CaM was bound to the C-terminal lobe of CaM. Near-UV CD spectra also indicated that the Trp of the peptide and Phe residues of the protein are involved in the binding. These results show that the CaM-binding domain of CaM kinase I binds to CaM in a manner analogous to that of myosin light chain kinase (AU)
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina , Calmodulina , Peptídeos , Dicroísmo CircularRESUMO
The interaction of an extracellular agonist (First Messenger) with its plasma membrane receptor leads to the transmission of a signal across the cell membrane and results in the production and or activation of other signalling molecules (Second Messengers). These Second Messengers control the action of many protein kinases and protein phosphatases and so lead to cellular responses. Although the biochemical basis of the transduction of signals in the main signalling systems in eukaryotic cells is probably largely known, intensified research is ongoing in the following areas: the discovery of specific substrates for many protein kinases, elucidation of the biological significance of the differential tissue expression and heterogeneity of many signalling proteins, and the unravelling of diverse interactions (such as signal potentiation, synergism, antagonism and neuronal co-transmission) between signalling systems. As knowledge from such studies accumulates, it is becoming clear that the 'cross talk' interactions between signalling systems are important features of dynamic cell regulation. This special issue is designed to summarize some aspects of the current work on various Second Messenger Systems and the integration of signals with respect to plasma membrane receptors, Second Messenger generation and degradation, protein kinases and phosphatases, cell cycle control, and cellular learning and mememory (AU)
Assuntos
Ratos , Humanos , Bioquímica , Sistemas do Segundo Mensageiro , Congresso/tendênciasRESUMO
Many short-lived proteins which are devoid of proteolytic activity contain PEST sequences which are segments along polypeptide chain that are rich in proline (P), glutamate (E), serine (S) and threonine (T). These designated PEST sequences are believed to be putative intramolecular signals for rapid proteolytic degradation. Calmodulin is a ubiquitous, 17 kDa, acidic Ca2+-binding protein which plays an important role in the regulation of many physiological processes through its interaction with a wide range of calmodulin-building proteins. Several calmodulin-binding proteins are known to contain PEST sequences and are susceptible to proteolysis by endogenous neutral proteases such as calpain I and calpain II. In this report, we discuss the functions of PEST sequences in calmodulin-binding proteins and assess the correlation between calmodulin-binding proteins and PEST sequences (AU)
Assuntos
Coelhos , Humanos , Proteínas de Ligação a Calmodulina , Prolina , Ácido Glutâmico , Serina , Treonina , Calpaína , UbiquitinaRESUMO
A high molecular weight calmodulin-binding protein (HMW CaMBP) from bovine heart cystosolic fraction was purified to apparent homogeneity. A novel CaM-dependent protein kinase was originally discovered when the total CaM-binding protein fraction from cardiac muscle was loaded on a gel filtration column. The CaM-dependent protein kinase has been highly purified by sequential chromatography on DEAE-Sepharose CI 6B (to remove calmodulin), CaM-Sepharose 4B, phosphocellulose, Sepharose 6B gel filtration and Mono S column chromatographies. The highly purified protein kinase stoichiometrically phosphorylated the HMW CaMBP in a Ca2+/CaM-dependent manner. The phosphorylation resulted in the maximal incorporation of 1 mol of phosphate/mol of the HMW CaMBP. The distinct substrate specificity of this protein kinase indicates that it is not related to the known protein kinases (I, II, III, IV and V) that have been already characterized, therefore we would like to designate this novel kinase as a CaM-dependent protein kinase VI (AU)