Interacting Protein Domains: Their Role in Signal and Energy Transduction (Nato ASI Subseries H:, 102)

This is now the fourth time that protein phosphorylation has been the focus of a NATO Advanced Study Institute. The first meeting with the topic "Signal Trans­ duction and Protein Phosphorylation" was held on the island of Spezai, Greece, in September 1986. The second one took place in Chateau La Londe, France, in September 1989 on "Cellular Regulation by Protein Phosphorylation", the third one on " Tyrosine Phosphorylation/Dephosphorylation and Downstream Signaling" was in September 1992 in Maratea, Italy. The titles of these books clearly mirror the developments that have taken place in the last decade. Beginning with the recognition that protein phosphorylation is at the center of signaling -clearly established in 1990 -it became apparent that many cellular processes are regulated by this mode. A new focus then emerged when it was recognized that growth factors are bound to corresponding receptors trigger protein tyrosine phosphorylation which controls cell prolifera­ tion. This was the topic of the third meeting in this series. It is now evident that further progress depends on understanding the three dimensional structure of the proteins involved. It goes without saying, for example, that understanding the location of proteins by adaptor proteins is only possible on the basis of the three dimensional protein structure. Therefore, the fourth meeting in this series concentrated on the protein structure of signaling molecules as well as on the elucidation of the principles of protein domain interactions. Front Matter....Pages I-X Front Matter....Pages 1-1 Control of Cellular Processes by Reversible Protein Phosphorylation....Pages 3-8 From Phosphorylase to Phosphorylase Kinase....Pages 9-14 Protein Kinase X: A Novel Human Protein Kinase closely related to the Catalytic Subunit of cAMP-dependent Protein Kinase....Pages 15-18 Interaction of Protein Kinase A c from Ascaris suum with Proteins and Peptides: Comparison with the Mammalian Enzyme....Pages 19-24 Front Matter....Pages 25-25 Interaction Studies Using Biosensors....Pages 27-36 Advances in Determination of Protein Structure by X-ray Diffraction Methods....Pages 37-44 Spectroscopical studies on the interacti on between ww domain and proline-rich peptides ....Pages 45-48 Novel Microscope-Based Approaches for the Investigation of Protein-Protein Interactions in Signal Transduction....Pages 49-52 Binding studies with SH2 domains from the phosphotyrosine kinase ZAP70 using surface plasmon resonance and scintillation proximity assays....Pages 53-57 Leucine Zipper mediated Homodimerization of Autoantigen L7 analyzed by Electrospray Ionization Mass Spectrometry and Yeast Two Hybrid Interactions....Pages 59-62 Proteins at Work: Time-Resolved FTIR Studies of Bacteriorhodopsin and the GTP-Binding Protein P21....Pages 63-72 Front Matter....Pages 73-73 Downstream Signaling from Phosphoinositide 3-Kinase....Pages 75-81 Phosphoinositide-3-Kinase Mediated Activation of JNK by the ßPDGFR....Pages 83-86 Regulation of Phospholipase C isozymes....Pages 87-92 Substrate Binding and Catalytic Mechanism in Phospholipase C from Bacillus cereus ....Pages 93-96 Identification of Three Active Site Residues Involved in Substrate Binding by Human 43 kDa-D- myo -Inositol 1,4,5- Tris phosphate 5-Phosphatase....Pages 97-100 Structural basis of protein-ligand interactions in the Pleckstrin Homology domain....Pages 101-104 Front Matter....Pages 105-105 Cell Signaling by Tyrosine Phosphorylation: The Other Side of the Coin....Pages 107-112 Binding studies on the neuronal isoform of the non-receptor protein tyrosine kinase pp60 c-src , pp60 c-srcN and potential target proteins....Pages 113-116 The switch cycle of the Ras protein and its role in signal transduction....Pages 117-138 Front Matter....Pages 105-105 In vivo quantitative assessment of Ras/Raf interaction using the two-hybrid system....Pages 139-142 in vivo Analysis of C-Raf1 — 14-3-3 Interaction....Pages 143-146 Signal Transduction through the MAP kinase Pathway....Pages 147-151 Identification and Characterization of MKKX, a novel mammalian MAP kinase kinase....Pages 153-156 Interleukin-1 activates a novel p54 MAP Kinase Kinase in rabbit liver....Pages 157-160 The Protein Kinase B Family — Structure, Regulation and Function....Pages 161-170 Regulation of p70 s6k ....Pages 171-185 Rapamycin, FRAP and the Control of 5’TOP mRNA Translation....Pages 187-194 Structure, Regulation and Targeting of Protein Phosphatase 2A....Pages 195-206 The Constitutive Activation of MET, RON and SEA Genes Induces Different Biological Responses....Pages 207-212 Nucleoside Diphosphate Kinase: Effect of the P100S Mutation on Activity and Quaternary Structure....Pages 213-216 Front Matter....Pages 217-217 Interaction of adenylyl cyclase type 1 with βγ subunits of heterotrimeric G-proteins....Pages 219-222 Regulation of G-protein Activation in Retinal Rods by Phosducin....Pages 223-226 Selective interactions of the rat μ-opioid receptor and a chimeric μ-opioid receptor expressed in COS-7 cells with multiple G proteins....Pages 227-230 Structural requirements for the EF-Tu-directed kinase....Pages 231-234 Effect of the Phytotoxin Fusicoccin on Plant Plasma Membrane H + -ATPase Expressed in Yeast....Pages 235-238 Organization and putative regulation of the glucose-specific phosphotransferase system from Staphylococcus carnosus ....Pages 239-244 Front Matter....Pages 245-245 Single Molecule Myosin Mechanics Measured Using Optical Trapping....Pages 247-259 Studies on the ATP-binding Site of Actin Using Site-directed Mutagenesis....Pages 261-264 Characterisation of the actin-binding protein Insertin....Pages 265-268 Front Matter....Pages 245-245 Cardiac Troponin....Pages 269-279 Studies on the function of the different phosphoforms of cardiac troponin I....Pages 281-284 Back Matter....Pages 287-292 The recently isolated gene PRKX encodes a novel type of human protein kinase closely related to the cAMP-dependent protein kinase (cAPK). The hydrophilic protein of about 41 kDa is called Protein Kinase X (Prkx) because the gene is located on the short arm of the human X-chromosome (Klink et al. 1995). It has a striking sequence similarity to the DC2 protein kinase from Drosophila melanogaster (62.3% identity in the conserved catalytic core region) and has a lower similarity to the catalytic subunits of lower organisms like that of Ascaris suum (53.5% identity) and Caenorhabditis elegans (51.1%). These protein kinases differ from the “classical” CAMP-dependent protein kinases (cAPKs) by their isoelectric points and the lengths of their branches in the phylogenetic tree (table 1, figure 1). Most residues important for substrate recognition and binding of the regulatory subunits RI and RII are identical comparing the catalytic subunit of cAMP-dependent protein kinase alpha (Adams and Taylor 1993; Grant et a1.1996) and Protein Kinase X. The core region of the kinase is also highly conserved whereas the N-terminus is totally different. The N-terminus of Protein Kinase X contains a putative binding motif for WW-domains, which is a proline-rich region called PY-motif (Macias et al. 1996), that might be important for regulation or subcellular localization. Northern blot analysis with different subfragments, of cDNA clones indicates a widespread expression with the highest level of expression observed in fetal and adult brain, kidney and lung and low levels of expression in all other tested tissues (Klink et al. 1995). Prkx was expressed in E. coli using several different expression systems. The understanding of cellular signaling - in eukaryotes mainly carried out by reversible protein phosphorylation - depends on knowledge of the three-dimensional structure of signaling molecules, i.e. proteins. Many aspects of studying proteins at the atomic level are discussed, e.g. advances in the determination of protein structure by X-ray diffraction methods, spectroscopical studies, and time-resolved Fourier transform (FTIR) spectroscopy. An area in which a quantum step in the development has occurred is the characterization of a single motor molecule, myosin. For the first time a completely new method - the application of laser traps - is available to assay the activity of a single motor molecule. A discussion of these methods is included in this book.