Regulatory phosphorylates

Muscle glycogen phosphorylase is a dimer of two identical subunits (842 residues, 97.44 kD). Each subunit contains a pyridoxal phosphate cofactor, covalently linked as a Schiff base to Lys °. Each subunit contains an active site (at the center of the subunit) and an allosteric effector site near the subunit interface (Eigure 15.15). In addition, a regulatory phosphorylation site is located at Ser on each subunit. A glycogen-binding site on each subunit facilitates prior association of glycogen phosphorylase with its substrate and also exerts regulatory control on the enzymatic reaction. 

GPCR function has been shown to be regulated by several different mechanisms. The number of receptors on the plasma membrane may be regulated by transcription, mRNA stability, biosynthetic processing, and protein stability. In addition, the function of receptors in the plasma membrane can be influenced by regulatory phosphorylation and by association with other proteins that determine the subcellular location of receptors relative to other signaling molecules. 

Norbury, C., Blow, J., and Nurse, P. (1991). Regulatory phosphorylation of the p34cdc2 protein kinase in vertebrates. EMBO J. 11 3321-3329. 

Pickham, K. M., Meyer, A. N., Li, J and Donoghue, D. J. (1992). Requirement of mosx protein kinase for meiotic maturation of Xenopus oocytes induced by a cdc2 mutant lacking regulatory phosphorylation sites. Mol. Cell. Biol. 12 3192-3203. 

Hentschel, P, Krucker, M., Grynhaum, M. D., Putzhach, K., Bischoff, R., and Albert, K., Determination of regulatory phosphorylation sites in nanogram amounts of a synthetic fragment of ZAP-70 using microprohe NMR and on-hne coupled capillary HPLC-NMR, Magnetic Resonance in Chemistry 43(9), 747-754, 2005. 

Ser and Thr residues that serve as regulatory phosphorylation sites. Mutations are also described for these regions leading to oncogenic activation of Raf kinase. The protein kinase activity is found in the CR3 domain. 

Munday, M.R. Campbell, D.G. Carling, D. Hardie, D.G. Identification by amino acid sequencing of three major regulatory phosphorylation sites on rat acetyl-CoA carboxylase. Eur. J. Biochem., 175, 331-338 (1988)... 

Jiao, J. Chollet, R. (1990). Regulatory phosphorylation of serine-15 in maize phosphoeno/pyruvate carboxylase by a C4-leaf protein-serine kinase. Archives of Biochemistry and Biophysics 283, 300-5. 

Jiao, J., Vidal, J., Echevarria, C. Chollet, R. (1991). In vivo regulatory phosphorylation site in C4-leaf phosphoeno/pyruvate carboxylase from maize and sorghum. Plant Physiology 96, 297-301. 

Obermann, W. M., Gautel, M., Weber, K., and Furst, D. O. (1997). Molecular structure of the sarcomeric M band Mapping of titin and myosin binding domains in myomesin and the identification of a potential regulatory phosphorylation site in myomesin. EMBOJ. 16, 211-220. 

RD Chen, JA Grobler, JH Hurley, AM Dean (1996). Second-site suppression of regulatory phosphorylation in Escherichia coli isocitrate dehydrogenase. Protein Sci 5 287-295, 1996. 

Douglas, P., Morrice, N., and MacKintosh, C., 1995, Identification of a regulatory phosphorylation site in the hinge 1 region of nitrate reductase from spinach (Spinacea oleracea) leaves, FEES Lett. 177 11311117. 

In addition to the receptors themselves, the nuclear receptor coactivators have also been found to be targets of regulatory phosphorylation events. 

These observations and mutation studies have shown that the activity of c-Src kinase is subject to strict regulation. The domain structure of c-Src kinase is shown in Fig. 8.17a. Src kinase carries a myristinic acid residue as a membrane anchor and harbors an SH2 and an SH3 domain N-terminal to its kinase domain. Furthermore, Src kinase possesses two important regulatory phosphorylation sites, namely Tyr 416 in the activation loop and Tyr 527 near the C-terminus. 

The y-isoenzyme lacks the C2 domain, is prenylated at the C-terminus, and lacks the regulatory phosphorylation sites of cPLAja. The enzyme demonstrates high lysophospho-lipase activity and lacks the acyl chain selectivity of the a-isoenzyme suggesting a role in phospholipid remodeling [23]. The 6, e, and -isoezymes have been identified more recently (H. Chiba, 2004 T. Ohto, 2005) and are discussed [23]. The group IV isoenzymes (a- ) have also been designated A-F [15]. 

Chapter 1, this volume). Phosphorylation is also essential for movement of actin filaments in both the Nitella and the sliding actin in vitro motility assays (Umemoto and Sellers, 1990 Warshaw etai, 1990 Sellers etal., 1985). The site of the regulatory phosphorylation is Ser-19 on the LC20 (Pearson et al., 1984). Myosin light chain kinase (MLCK) also phosphorylates Thr-18, albeit at a much lower rate (Ikebe et al., 1986). Thr-18 phosphorylation increases the actin-activated MgATPase activity (Ikebe et al., 1988), but does not increase the rate of actin filament sliding in either of the two motility assays (Sellers et al., 1985 Okagaki et al, 1991). 

When in the presence of endogenous currents, the presence or absence of subunits (directly associated regulatory proteins) and kinases or phosphatases controlling regulatory phosphorylation sites can affect the pharmacology of the expressed channel protein relative to native cardiac ion channels. 

As in the case of the dikinase regulatory cascade (see Fig. 1), little is known with certainty with respect to how light modulates the phosphorylation-status (activation-state) of cytoplasmic PEPC other than it, too, is dependent, either directly or indirectly, on photosynthetic electron transport and/or photophosphorylation (19, 20). Neither dithiothreitol-reduced spinach leaf thioredoxin h [Td (30)], PPi, fructose 2,6-bisphosphate (Fru 2,6-P2), various combinations of EGTA/calcium/calmodulin (CaM) [see Table 1 (Refs. 29, 31)] nor the CaM-antagonist calmidazolium (R. Chollet and J. Vidal, unpublished) has any significant effect on the regulatory phosphorylation and/or activation of purified dark-form PEPC in our reconstituted in vitro system. A one-hour preincubation of the dark-form PK with Mg-ATP is also without effect on its subsequent activation of PEPC. Thus, we currently have no supportive evidence... 

Stanford JS, Ruderman JV. 2005. Changes in regulatory phosphorylation of Cdc25C Ser287 and Weel Ser549 during normal cell cycle progression and checkpoint arrests. Mol Biol Cell 16(12) 5749-5760. 

Segment V is the variable, COOH-terminal portion of the molecule containing regulatory phosphorylation sites. The mammalian muscle isoform has 7 sites, the liver isoform 5 and the yeast Gsy2p 3 sites. Two of the yeast sites can readily be seen as conserved versions of mammalian sites 3a and 3b. It is possible that the yeast and mammalian enzymes share some phosphorylation controls but not others. 

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