Eukaryotes phosphorylation

This posttranscriptional modification occurs in more than 30% of proteins in mammals. This involves the addition of one or more PO4 groups to particular amino acids in a protein. In mammalian cells, PO4 groups are added to the amino acids threonine, serine, or tyrosine of the protein. In contrast, amino acids such as aspartic, glutamic, and histidine are phosphorylated in prokaryotes, instead of tyrosine, serine, and threonine in eukaryotes. Occasionally in both prokaryotes and eukaryotes, phosphorylation occurs in arginine, lysine, and cystein residues of the protein. The ratio of phosphorylation of the three amino acid residues in mammalian cells is 1000 100 1 for threonine, serine, and tyrosine. Proteins are phosphorylated at more than one site, and usually a mixture of phosphorylated isomers with different levels of phosphorylation exists in the cell. Phosphorylation adds a negative charge to the proteins with the addition of the PC>4 group. 

FIGURE 20.1 Pyruvate produced hi glycolysis is oxidized in the tricarboxylic acid (TCA) cycle. Electrons liberated in this oxidation flow through the electron transport chain and drive the synthesis of ATP in oxidative phosphorylation. In eukaryotic cells, this overall process occurs in mitochondria. 

The processes of electron transport and oxidative phosphorylation are membrane-associated. Bacteria are the simplest life form, and bacterial cells typically consist of a single cellular compartment surrounded by a plasma membrane and a more rigid cell wall. In such a system, the conversion of energy from NADH and [FADHg] to the energy of ATP via electron transport and oxidative phosphorylation is carried out at (and across) the plasma membrane. In eukaryotic cells, electron transport and oxidative phosphorylation are localized in mitochondria, which are also the sites of TCA cycle activity and (as we shall see in Chapter 24) fatty acid oxidation. Mammalian cells contain from 800 to 2500 mitochondria other types of cells may have as few as one or two or as many as half a million mitochondria. Human erythrocytes, whose purpose is simply to transport oxygen to tissues, contain no mitochondria at all. The typical mitochondrion is about 0.5 0.3 microns in diameter and from 0.5 micron to several microns long its overall shape is sensitive to metabolic conditions in the cell. 

Figure 1. The cell cycle as a Cdc2 cycle. Progression through the eukaryotic cell cycle is sensitive to the phosphorylation state of Cdc2. A block to DNA synthesis (S) prevents dephosphorylation, and hence activation, of Cdc2. Impaired spindle function will prevent deactivation of Cdc2 and thus blocks exit from M phase (Hoyt et al., 1991 Li and Murray, 1991 reviewed in Nurse, 1991). Exit from M phase requires destruction of the regulatory subunit, Cyc B. Dephosphorylation of Cdc2 at thr-161 may act to destabilize the Cdc2/Cyc B complex and thus allow the ubiquitination of Cyc B followed by its destruction.

Post-translational modification of proteins plays a critical role in cellular function. For, example protein phosphorylation events control the majority of the signal transduction pathways in eukaryotic cells. Therefore, an important goal of proteomics is the identification of post-translational modifications. Proteins can undergo a wide range of post-translational modifications such as phosphorylation, glycosylation, sulphonation, palmitoylation and ADP-ribosylation. These modifications can play an essential role in the function of the protein and mass spectrometry has been used to characterize such modifications. 

Kimball, S. R., Fabian, J. R., Pavitt, G. D., Hinnebusch, A. G., and Jefferson, L. S. (1998). Regulation of guanine nucleotide exchange through phosphorylation of eukaryotic initiation factor eIF2alpha. Role of the alpha- and delta-subunits of eIF2B. J. Biol. Chem. 273, 12841-12845. 

Matts, R. L., Levin, D. H., and London, I. M. (1983). Effect of phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 on the function of reversing factor in the initiation of protein synthesis. Proc. Natl. Acad. Sci. USA 80, 2559—2563. 

Panniers, R., and Henshaw, E. C. (1983). A GDP/GTP exchange factor essential for eukaryotic initiation factor 2 cycling in Ehrlich ascites tumor cells and its regulation by eukaryotic initiation factor 2 phosphorylation. J. Biol. Chem. 258, 7928—7934. 

Wang, X., Paulin, F. E., Campbell, L. E., Gomez, E., O Brien, K., Morrice, N., and Proud, C. G. (2001). Eukaryotic initiation factor 2B Identification of multiple phosphorylation sites in the epsilon-subunit and their functions in vivo. EMBO J. 20,... 

Mazroui, R., Sukarieh, R., Bordeleau, M. E., Kaufman, R. J., Northcote, P., Tanaka, J., Gallouzi, I., and Pelletier, J. (2006). Inhibition of ribosome recruitment induces stress granule formation independendy of eukaryotic initiation factor 2alpha phosphorylation. Mol. Biol. Cell 17, 4212-4219. 

McEwen, E., Kedersha, N., Song, B., Scheuner, D., Gilks, N., Han, A., Chen, J. J., Anderson, P., and Kaufman, R. J. (2005). Heme-regulated inhibitor (HRI) kinase-mediated phosphorylation of eukaryotic translation initiation factor 2 (eIF2) inhibits translation, induces stress granule formation, and mediates survival upon arsenite exposure. J. Biol. Chem. 280, 16925—16933. 

Ueda, T., Watanabe-Fukunaga, R., Fukuyama, H., Nagata, S., and Fukunaga, R. (2004). Mnk2 and Mnkl are essential for constitutive and inducible phosphorylation of eukaryotic initiation factor 4E but not for cell growth or development. Mol. Cell Biol. 24, 6539-6549. 

Wang, X., Flynn, A., Waskiewicz, A. J., Webb, B. L. J., Vries, R. G., Baines, I. A., Cooper, J., and Proud, C. G. (1998). The phosphorylation of eukaryotic initiation factor eIF4E in response to phorbol esters, cell stresses, and cytokines is mediated by distinct MAP kinase pathways. J. Biol. Chem. 273, 9373-9377. 

Waskiewicz, A. J., Johnson, J. C., Penn, B., Mahalingam, M., Kimball, S. R., and Cooper, J. A. (1999). Phosphorylation of the cap-binding protein eukaryotic translation factor 4E by protein kinase Mnkl in vivo. Mol. Cell. Biol. 19, 1871—1880. 

Figure 13.1 Schematic diagram of eukaryotic translation initiation. The sites of action of small molecule inhibitors are shown with dashed lines. Kinases that affect the phosphorylation of 4E-BP and eIF2a, and exert effects on ribosome recruitment and ternary complex formation, respectively, are shown in a black box. See text for details.

Human eukaryotic translation initiation factor 4G (eIF4G) recruits mnkl to phosphorylate eIF4E. EMBO J. 18, 270-279. 

Mitogen-activated protein kinase phosphatases are dual-function protein phosphatases. Just as the MAPK kinases (e.g. MEKs) are unique as dual-functioning kinases in that they phosphorylate MAPKs on threonine and tyrosine residues, there are unique dual-function ing protein phosphatases that reverse the phosphorylation and activation of MAPKs [43], Such MAPK phosphatases (MKPs) were first identified as a product of vaccinia virus (VH1) and later found in all eukaryotic cells. There are now numerous members of this VH1 family of dual-functioning protein phosphatases. 

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