A glycine-rich loop in the ATP-binding site is one of the most highly conserved sequence motifs in protein kinases. Each conserved glycine (Gly-50, Gly-52, and Gly-55) in the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) was replaced with Ser and/or Ala. Active mutant proteins were expressed in Escherichia coli, purified to apparent homogeneity, separated into phosphoisoforms, and characterized. Replacing Gly-55 had minimal effects on steady-state kinetic parameters, whereas replacement of either Gly-50 or Gly-52 had major effects on both K m and k cat values consistent with the prediction of the importance of the tip of the glycinerich loop for catalysis. Substitution of Gly-50 caused a 5-8-fold reduction in K m (ATP) , an 8 -12-fold increase in K m (peptide) , and a 3-5-fold drop in k cat . The K m (ATP) and K m (peptide) values of C(G52S) were increased 8-and 18-fold, respectively, and the k cat was decreased 6-fold. In contrast to catalytic efficiency, the ATPase rates of C(G50S) and C(G52S) were increased by more than an order of magnitude. The thermostability of each mutant was slightly increased. Unphosphorylated C(G52S) was characterized as well as several isoforms phosphorylated at a single site, Ser-338. All of these phosphorylation-defective mutants displayed a substantial decrease in both enzymatic activity and thermal stability that correlated with the missing phosphate at Thr-197. These results are correlated with the crystal structure, models of the respective mutant proteins, and conservation of the Glys within the protein kinase family.The eukaryotic protein kinases specific for serine, threonine, and tyrosine all share a conserved catalytic core that folds into a topologically similar three-dimensional structure (2-5). Particularly conserved in this core are the amino acids involved in nucleotide binding and catalysis with one of the most highly conserved features being a GXGXXG motif near the amino terminus. The first and second glycines of this triad are essentially invariant, whereas the third is somewhat more variable but is always a small residue. Our goal is to understand the functional importance of this motif, a hallmark of the protein kinase family.The catalytic (C) 1 subunit of cAMP-dependent protein kinase (cAPK), one of the most extensively investigated members of the protein kinase family, has served repeatedly as a prototype for studying structure-function relationships in this enzyme family (6, 7). It is a relatively simple protein kinase, and both its regulatory and catalytic subunits can be purified readily as active proteins in Escherichia coli. The glycine-rich motif in the C-subunit, (LG 50 TG 52 SFG 55 RV), displays at each position the amino acid residue found in the majority of protein kinases (3, 4). The first crystal structure of a protein kinase was a binary complex of the C-subunit and a peptide inhibitor (2, 8). Based on this structure and on subsequent structures of other protein kinases, the conserved features of the unique protein kinase nucleotide...

Over the past years, a concept for creatine kinase function, the 'PCr-circuit' model, has evolved. Based on this concept, multiple functions for the CK/PCr-system have been proposed, such as an energy buffering function, regulatory functions, as well as an energy transport function, mostly based on studies with muscle. While the temporal energy buffering and metabolic regulatory roles of CK are widely accepted, the spatial buffering or energy transport function, that is, the shuttling of PCr and Cr between sites of energy utilization and energy demand, is still being debated. There is, however, much circumstantial evidence, that supports the latter role of CK including the distinct, isoenzyme-specific subcellular localization of CK isoenzymes, the isolation and characterization of functionally coupled in vitro microcompartments of CK with a variety of cellular ATPases, and the observed functional coupling of mitochondrial oxidative phosphorylation with mitochondrial CK. New insight concerning the functions of the CK/PCr-system has been gained from recent M-CK null-mutant transgenic mice and by the investigation of CK localization and function in certain highly specialized non-muscle tissues and cells, such as electrocytes, retina photoreceptor cells, brain cells, kidney, salt glands, myometrium, placenta, pancreas, thymus, thyroid, intestinal brush-border epithelial cells, endothelial cells, cartilage and bone cells, macrophages, blood platelets, tumor and cancer cells. Studies with electric organ, including in vivo 31p_ NMR, clearly reveal the buffer function of the CK/PCr-system in electrocytes and additionally corroborate a direct functional coupling of membrane-bound CK to the Na+/K+-ATPase. On the other hand, experiments with live sperm and recent in vivo 31p-NMR measurements on brain provide convincing evidence for the transport function of the CK/PCr-system. We report on new findings concerning the isoenzyme-specific cellular localization and subcellular compartmentation of CK isoenzymes in photoreceptor cells, in glial and neuronal cells of the cerebellum and in spermatozoa. Finally, the regulation of CK expression by hormones is discussed, and new developments concerning a connection of CK with malignancy and cancer are illuminated. Most interesting in this respect is the observed upregulation of CK expression by adenoviral oncogenes. (Mol Cell Biochem 133/134: 193-220,1994) Key words: creatine kinase, functional coupling with cellular ATPases, spermatozoa, electrocytes, retina, cerebellum Abbreviations: M-CK, B-CK and Mi-CK refer to muscle-type, brain-type and mitochondrial-type creatine kinase, respectively, with the cytosolic isoforms MM-, MB-and BB-CK forming dimers and Mi-CK forming dimers as well as octamers; BGC -Bergmann

Over the past years, a concept for creatine kinase function, the 'PCr-circuit' model, has evolved. Based on this concept, multiple functions for the CK/PCr-system have been proposed, such as an energy buffering function, regulatory functions, as well as an energy transport function, mostly based on studies with muscle. While the temporal energy buffering and metabolic regulatory roles of CK are widely accepted, the spatial buffering or energy transport function, that is, the shuttling of PCr and Cr between sites of energy utilization and energy demand, is still being debated. There is, however, much circumstantial evidence, that supports the latter role of CK including the distinct, isoenzyme-specific subcellular localization of CK isoenzymes, the isolation and characterization of functionally coupled in vitro microcompartments of CK with a variety of cellular ATPases, and the observed functional coupling of mitochondrial oxidative phosphorylation with mitochondrial CK. New insight concerning the functions of the CK/PCr-system has been gained from recent M-CK null-mutant transgenic mice and by the investigation of CK localization and function in certain highly specialized non-muscle tissues and cells, such as electrocytes, retina photoreceptor cells, brain cells, kidney, salt glands, myometrium, placenta, pancreas, thymus, thyroid, intestinal brush-border epithelial cells, endothelial cells, cartilage and bone cells, macrophages, blood platelets, tumor and cancer cells. Studies with electric organ, including in vivo 31P-NMR, clearly reveal the buffer function of the CK/PCr-system in electrocytes and additionally corroborate a direct functional coupling of membrane-bound CK to the Na+/K(+)-ATPase. On the other hand, experiments with live sperm and recent in vivo 31P-NMR measurements on brain provide convincing evidence for the transport function of the CK/PCr-system. We report on new findings concerning the isoenzyme-specific cellular localization and subcellular compartmentation of CK isoenzymes in photoreceptor cells, in glial and neuronal cells of the cerebellum and in spermatozoa. Finally, the regulation of CK expression by hormones is discussed, and new developments concerning a connection of CK with malignancy and cancer are illuminated. Most interesting in this respect is the observed upregulation of CK expression by adenoviral oncogenes.

The distinct isoenzyme-specific localization of creatine kinase (CK) isoenzymes found recently in brain suggests an important function for CK in brain energetics and points to adaptation of the CK system to the special energy requirements of different neuronal and glial cell types. For example, the presence of brain-type B-CK in Bergmann glial cells and astrocytes is very likely related to the energy requirements for ion homeostasis (K⁺-resorption) in the brain, as well as for metabolite and neurotransmitter trafficking between glial cells and neurons. In contrast, the presence of muscle-type M-CK, found exclusively in Purkinje neurons which also express other muscle-specific protein, is very likely related to the unique calcium metabolism of these neurons. In addition, the developmentally late appearance of mitochondrial CK (Mi-CK) during brain development indicates an important function for Mi-CK in the oxidative energy metabolism of the brain. The physiological importance of the phosphocreatine circuit fully operating in adult brain has been corroborated by recent data from in vivo ³¹P-NMR magnetization transfer measurements. Future investigations should concentrate on the possible involvement of CK in diseases of the CNS with altered energy metabolism, aspects of which are also discussed here.

The conserved glycines in the glycine-rich loop (Leu-Gly 50 -Thr-Gly 52 -Ser-Phe-Gly 55 -ArgVal) of the catalytic (C) subunit of cAMP-dependent protein kinase were each mutated to Ser (G50S, G52S, and G55S). The effects of these mutations were assessed here using both steady-state and presteady-state kinetic methods. While G50S and G52S reduced the apparent affinity for ATP by approximately 10-fold, substitution at Gly55 had no effect on nucleotide binding. In contrast to ATP, only mutation at position 50 interfered with ADP binding. These three mutations lowered the rate of phosphoryl transfer by 7-300-fold. The combined data indicate that G50 and G52 are the most critical residues in the loop for catalysis, with replacement at position 52 being the most extreme owing to a larger decrease in the rate of phosphoryl transfer (29 vs 1.6 s -1 in contrast to 500 s -1 for wild-type C). Surprisingly, all three mutations lowered the affinity for Kemptide by approximately 10-fold, although none of the loop glycines makes direct contact with the substrate. The inability to correlate the rate constant for net product release with the dissociation constant for ADP implies that other steps may limit the decomposition of the ternary product complex. The observations that G52S (a) selectively affects ATP binding and (b) significantly lowers the rate of phosphoryl transfer without making direct contact with either the nucleotide or the peptide imply that this residue serves a structural role in the loop, most likely by positioning the backbone amide of Ser53 for contacting the γ-phosphate of ATP. Energyminimized models of the mutant proteins are consistent with the observed kinetic consequences of each mutation. The models predict that only mutation of Gly52 will interfere with the observed hydrogen bonding between the backbone and ATP.Many nucleotide binding enzymes utilize glycine-rich loops that help position nucleotides for catalysis (1-3). Crystal structures of dinucleotide and mononucleotide binding enzymes define several glycine-rich loops that are distinct both in their amino acid sequence and in their orientation relative to the nucleotide. The glycine-rich loops found in dinucleotide binding enzymes [e.g., pyruvate dehydrogenase (4) and dihydrofolate reductase (5)] are typically Gly-X-Gly-X-X-Gly and provide space for the dinucleotide pyrophosphate moiety. The glycine-rich loops, or P-loops, found in mononucleotide binding enzymes [e.g., p21ras (6), adenylate kinase (7), Rec A (8), and elongation factor Tu (9)] have the consensus sequence Gly-X-X-X-X-Gly-Lys-Ser/Thr and interact with the γ-phosphate of ATP (10). In contrast, the protein kinase family of enzymes contain a glycine-rich loop with a consensus sequence Gly-X-Gly-X-X-Gly-X-Val. On the basis of numerous crystal structures, this loop is part of a -hairpin that connects -strands 1 and 2 and serves as a nucleotide-positioning loop (NPL) 1 that interacts with all three phosphates of ATP (11)(12)(13)(14)(15). The glycines in the protein kinase NPL are ...