We have used isotope-edited nuclear magnetic resonance spectroscopy, binding studies, and ATPase activity assays to investigate the interaction with F-actin of the 10 kDa C-terminal 658C fragment of chicken gizzard caldesmon and two site-directed mutants of this fragment. Simultaneous dual-sited contacts with F-actin are observed for the segments of the 658C sequence flanking tryptophan residues 692 and 722. Competition experiments showed that both 658C contacts with actin are displaced by substoichiometric concentrations of the short inhibitory region of troponin-I indicative of different binding sites on actin for these regions of troponin-I and caldesmon. Substitution of caldesmon serine-702 by aspartic acid within the spacer region linking the two actin contacts of 658C led to weaker binding but with retention of equivalent affinity for each interaction site. Differential binding affinity of the two sites was achieved by replacement of the sequence Glu691-Trp-Leu-Thr-Lys-Thr696 by Pro-Gly-His-Tyr-Asn-Asn. Consistent with these data, the concentration of this Cg1 mutant required to achieve 50% inhibition of actin-tropomyosin-activated myosin ATPase was 4-fold greater than found for the 658C fragment. Although calmodulin binding to Cg1 was observed, calmodulin proved ineffective in relieving the inhibition induced by the binding of this mutant to actin. These results are discussed in light of the actin contacts which are involved in the inhibitory activity possessed by different regions of the C-terminus of caldesmon.

The pattern of phosphorylation of adjacent serine residues in several peptides based on the N-terminal region of human cardiac troponin I has been analysed by PAGE and 1H NMR spectroscopy to identify the products. With cAMP-dependent protein kinase, Ser24 is rapidly phosphorylated, and subsequent much slower phosphorylation of Ser23 occurs only after phosphorylation of Ser24 is almost complete. Monophosphorylation of the peptide at Ser23 was not detected at any time. On replacement of Arg22 with Ala or Met the sole phosphorylation target was Ser23, phosphorylation being considerably slower than for Ser24 in the wild-type peptide, while diphosphorylation could not be detected after prolonged incubation. The results emphasise the importance of the N-terminal sequence RRRSS for the function of cardiac troponin I and imply that in human cardiac muscle unstimulated by adrenaline, troponin I is phosphorylated on Ser24. Comparative two-dimensional NOESY data indicate that in the diphosphorylated form at physiological pH values, specific structural constraints are imposed on the N-terminal peptide region. These constraints result in the effective screening of the two phosphate groups from each other by the arginine residues N-terminal to the serine pair and stabilisation of the structure in the region of residues 25-29, which is adjacent to a site of interaction between troponin I and troponin C. These conformational changes presumably underlie the decrease in calcium sensitivity of the myofibrillar ATPase that occurs after adrenaline intervention.

Conference Article| May 01 1995 Comparative phosphorylation kinetics of the human insulin receptor protein-tyrosine kinase domain and an enzymatically competent deletion construct - the tail could wag the dog PHILIP G. QUIRK; PHILIP G. QUIRK 1School of Biochemistry, University of Birmingham, Birmingham B15 2TT, U.K. Search for other works by this author on: This Site PubMed Google Scholar NOELEEN E. KEANE; NOELEEN E. KEANE 1School of Biochemistry, University of Birmingham, Birmingham B15 2TT, U.K. Search for other works by this author on: This Site PubMed Google Scholar BARRY A. LEVINE; BARRY A. LEVINE 1School of Biochemistry, University of Birmingham, Birmingham B15 2TT, U.K. Search for other works by this author on: This Site PubMed Google Scholar LEI WEI; LEI WEI *W.M. Keck Center for Genome Informatics, Institute for Biosciences and Technology, Texas A & M University, Houston, TX 77030, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar LELAND ELLIS LELAND ELLIS *W.M. Keck Center for Genome Informatics, Institute for Biosciences and Technology, Texas A & M University, Houston, TX 77030, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1995) 23 (2): 188S. https://doi.org/10.1042/bst023188s Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Cite Icon Cite Get Permissions Citation PHILIP G. QUIRK, NOELEEN E. KEANE, BARRY A. LEVINE, LEI WEI, LELAND ELLIS; Comparative phosphorylation kinetics of the human insulin receptor protein-tyrosine kinase domain and an enzymatically competent deletion construct - the tail could wag the dog. Biochem Soc Trans 1 May 1995; 23 (2): 188S. doi: https://doi.org/10.1042/bst023188s Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search © 1995 Biochemical Society1995 Article PDF first page preview Close Modal You do not currently have access to this content.

Conference Article| May 01 1995 A basic residue C-terminal to tyrosine compromises its viability as a tyrosine kinase target NOELEEN E. KEANE; NOELEEN E. KEANE 1NMR Unit, School of Biochemistry, University of Birmingham, Birmingham, B15 2TT U.K. Search for other works by this author on: This Site PubMed Google Scholar PHILIP G. QUIRK; PHILIP G. QUIRK 1NMR Unit, School of Biochemistry, University of Birmingham, Birmingham, B15 2TT U.K. Search for other works by this author on: This Site PubMed Google Scholar BARRY A. LEVINE; BARRY A. LEVINE 1NMR Unit, School of Biochemistry, University of Birmingham, Birmingham, B15 2TT U.K. Search for other works by this author on: This Site PubMed Google Scholar ALAIN CHAVANIEU; ALAIN CHAVANIEU *CNRS, Montpellier, France Search for other works by this author on: This Site PubMed Google Scholar BERNARD CALAS; BERNARD CALAS *CNRS, Montpellier, France Search for other works by this author on: This Site PubMed Google Scholar YUAN GAO; YUAN GAO 1NMR Unit, School of Biochemistry, University of Birmingham, Birmingham, B15 2TT U.K. Search for other works by this author on: This Site PubMed Google Scholar LEI WEI; LEI WEI ^W.M. Keck Center for Genome Informatics, IBT, Texas A & M University, Houston TX, USA Search for other works by this author on: This Site PubMed Google Scholar LELAND ELLIS LELAND ELLIS ^W.M. Keck Center for Genome Informatics, IBT, Texas A & M University, Houston TX, USA Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1995) 23 (2): 192S. https://doi.org/10.1042/bst023192s Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Cite Icon Cite Get Permissions Citation NOELEEN E. KEANE, PHILIP G. QUIRK, BARRY A. LEVINE, ALAIN CHAVANIEU, BERNARD CALAS, YUAN GAO, LEI WEI, LELAND ELLIS; A basic residue C-terminal to tyrosine compromises its viability as a tyrosine kinase target. Biochem Soc Trans 1 May 1995; 23 (2): 192S. doi: https://doi.org/10.1042/bst023192s Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1995 Biochemical Society1995 Article PDF first page preview Close Modal You do not currently have access to this content.

1H-NMR and 31P-NMR spectroscopy were employed to assess the electrostatic consequences of phosphorylation of single and multiple tyrosine residues in peptides derived from the core and tail autophosphorylation regions of the human insulin receptor tyrosine-kinase domain. In both peptides, phosphorylation was accompanied by changes in the resonances from basic side-chains; those from acidic residues were unaffected. Tyrosine phosphorylation caused increases of up to one in the pKa values of histidine residues situated up to eight residues away in the primary sequence. Titration curve analysis by Hill plots suggested some cooperativity of histidine and phosphate ionizations. Behaviour closely analogous to that of the insulin receptor tail peptide was observed during changes in phosphorylation of the intact insulin receptor kinase domain, suggesting that the electrostatic dissemination effects seen for the isolated peptide are retained by the peptide sequence in the context of the much larger protein. Similar changes in the behaviour of basic residues were also observed upon tyrosine phosphorylation of a cdc2-derived peptide, suggesting that this potential of phosphorylation events to propagate directed structural changes may find a widespread utility in the activation of protein kinases and in the transduction of phosphorylation-based signalling.

Using NMR spectroscopy to visualise tyrosine phosphorylation kinetics in real time, we have investigated the sequence-dependent determinants of the selectivity of the human insulin receptor protein-tyrosine kinase for different tyrosine residues. The peptides used encompass the multiple-tyrosine-containing autophosphorylation site sequences from the insulin receptor kinase core domain (Tyr1158, Tyr1162 and Tyr1163) and from its specific C-terminal tail domain (Tyr1328 and Tyr1334). Comparison of the phosphorylation kinetics with those found for the tyrosine residues on a peptide comprising the regulatory tyrosine phosphorylation site of cdc2 points to the role of the primary sequence context of the phosphate acceptor. The particularly deleterious influence of a basic residue immediately C-terminal to the tyrosine is discussed in relation to the autophosphorylation properties of the regulatory loop regions of the insulin and epidermal growth factor receptor kinases. The data further suggest that receptor tyrosine kinase active sites and their substrate targets act in concert to ensure that specific downstream effects are activated.

Conference Article| August 01 1993 Substrate specificity of the insulin receptor tyrosine kinase domain Noeleen E. Keane; Noeleen E. Keane 1School of Biochemistry, University of Birmingham, Birmingham B15 2TT U.K. Search for other works by this author on: This Site PubMed Google Scholar Barry A. Levine; Barry A. Levine 1School of Biochemistry, University of Birmingham, Birmingham B15 2TT U.K. Search for other works by this author on: This Site PubMed Google Scholar Philip Quirk; Philip Quirk 1School of Biochemistry, University of Birmingham, Birmingham B15 2TT U.K. Search for other works by this author on: This Site PubMed Google Scholar Bernard Calas; Bernard Calas 2CRNS, Montpellier, France Search for other works by this author on: This Site PubMed Google Scholar Alain Chavanieu; Alain Chavanieu 2CRNS, Montpellier, France Search for other works by this author on: This Site PubMed Google Scholar Florin Grigorescu; Florin Grigorescu 2CRNS, Montpellier, France Search for other works by this author on: This Site PubMed Google Scholar Leland Ellis Leland Ellis 3Institute of Biosciences and Technology, Texas A. & M. University, Houston, Texas, USA Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1993) 21 (3): 266S. https://doi.org/10.1042/bst021266s Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation Noeleen E. Keane, Barry A. Levine, Philip Quirk, Bernard Calas, Alain Chavanieu, Florin Grigorescu, Leland Ellis; Substrate specificity of the insulin receptor tyrosine kinase domain. Biochem Soc Trans 1 August 1993; 21 (3): 266S. doi: https://doi.org/10.1042/bst021266s Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu nav search search input Search input auto suggest search filter All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1993 Biochemical Society1993 Article PDF first page preview Close Modal You do not currently have access to this content.