Phosphorylation, of proteins

In mammalian cells, the two most common forms of covalent modification are partial proteolysis and ph osphorylation. Because cells lack the ability to reunite the two portions of a protein produced by hydrolysis of a peptide bond, proteolysis constitutes an irreversible modification. By contrast, phosphorylation is a reversible modification process. The phosphorylation of proteins on seryl, threonyl, or tyrosyl residues, catalyzed by protein kinases, is thermodynamically spontaneous. Equally spontaneous is the hydrolytic removal of these phosphoryl groups by enzymes called protein phosphatases. 

One main line of future research could be in the inhibitory/activating effect on key enzymes involved in the pathogenesis of arteriosclerosis. In particular, enzymes regulating signal transduction involved in phosphorylation of proteins, such as PKC and tyrosine protein kinase, seems to be somehow modulated by different polyphenols and may represent a possible target for polyphenol activity. 

Hunter, T., The Croonian Lecture 1997. The phosphorylation of proteins on tyrosine its role in cell growth and disease, Philos. Trans. Roy. Soc. London B (Biol. Sci.), 353, 583-605, 1998. 

Hormonal actions on target neurons are classified in terms of cellular mechanisms of action. Hormones act either via cell-surface or intracellular receptors. Peptide hormones and amino-acid derivatives, such as epinephrine, act on cell-surface receptors that do such things as open ion-channels, cause rapid electrical responses and facilitate exocytosis of hormones or neurotransmitters. Alternatively, they activate second-messenger systems at the cell membrane, such as those involving cAMP, Ca2+/ calmodulin or phosphoinositides (see Chs 20 and 24), which leads to phosphorylation of proteins inside various parts of the target cell (Fig. 52-2A). Steroid hormones and thyroid hormone, on the other hand, act on intracellular receptors in cell nuclei to regulate gene expression and protein synthesis (Fig. 52-2B). Steroid hormones can also affect cell-surface events via receptors at or near the cell surface. 

Okadaic acid is a powerful tumor promoter of a nonphorbol ester type that inhibits protein phosphatase-1 and -2A in vitro (Haystead et al., 1989). Okadaic acid directly stimulates smooth muscle contraction (Haystead et al., 1989) and probably causes diarrhea, either by stimulating the phosphorylation of proteins controlling sodium secretion by intestinal cells or by increasing phosphorylation of elements that regulate permeability to solutes, resulting in a passive loss of fluids (Aune and Yndestad, 1993). 

As might be expected from other mechanisms of regulation described in this text, phosphorylation and dephosphorylation of key proteins is the main mechanism for regulating the cycle, i.e. reversible phosphorylation, also known as interconversion cycles (discussed in Chapter 3). In the cell cycle, several of these interconversion cycles play a role in control at the checkpoints. Two important terms must be appreciated to help understand the mechanism of regulation of the cycle the phosphorylation of proteins is catalysed by specific protein kinases, known as cell-division kinases (cdck) or cell cycle kinases (cck) and these enzymes are activated by specific proteins, known as cyclins. 

Any of a broad class of phosphoryl-transfer enzymes [EC 2.7.1.x] that catalyze the ATP-dependent phosphorylation of proteins, most often occurring at seryl, threo-nyl, and tyrosyl residues. These enzymes are central participants in cellular signal transduction pathways, and their discovery and recognition as primary control components of the cell culminated in the award of the 1992 Nobel Prize in Medicine and Physiology to American enzymologists Edwin Krebs and Edward Fischer. There is reason to believe that approximately 2% of the coding sequences in the human genome specify some 2000 different kinases that phosphorylate protein substrates. The prototypical enzyme is known as 3, 5 -cAMP-stimulated protein kinase (or, protein kinase A). See specific protein kinase... 

Aromatic amino acids are biogenetic precursors of neuroamines (dopamine, serotonin, histamine, etc.). On the other hand, phenylalanine (Phe) is frequently present in peptide sequences, while tyrosine is an important site of phosphorylation of proteins. Aromatic amino acids and neuroamines fluorinated on the aromatic ring have been the focus of many investigations. Indeed, after incorporation in polypeptides and proteins, they can be used as probes in NMR and in PET. 

The phosphorylation of proteins on Ser, Thr or Tyr residues is a basic tool for the regulation of protein activity (see 7.1). Many eucaryotic transcriptional activators are isolated as phosphorylated proteins. The phosphorylation occurrs mainly on the Ser and Thr residues, but can also be observed on the Tyr residues. The extent of phosphorylation is regulated via specific protein kinases and protein phosphatases, each components of signal transduction pathways (see ch. 7). The phosphorylation of transcriptio-... 

A central tool for signal transmission in a cell is phosphorylation of proteins via protein kinases. Proteins can be reversibly activated or inactivated via phosphorylation. The phosphorylation status of a protein is controlled by the activity of both protein kinases and protein phosphatases (see chapter 7). Both classes of enzymes are elementary components of signaling pathways and their activity is subject to manifold regulation. 

The consensus sequence for phosphorylation of proteins by protein kinase A is RRXSX. The RII subrmit contains such a sequence in the autoinhibitory domain and is therefore subject to phosphorylation by the C subrmit in the holoenzyme, but without release of inhibition. Inhibition of the C subrmit by the R subrmit is based on binding of the autoinhibitory sequence of R at the substrate binding site and at parts of the active center of the C subrmit. 

Fig. 7.14. Regulation of CaM kinase II. Scheme of regulation of CaM kinase II by Ca Vcalmodu-lin and by autophosphorylation. CaM kinase II is inactive in the unphosphorylated form and in the absence of Ca calmodulin. Binding of Ca Vcalmodulin activates the kinase for phosphorylation of protein substrates. In the process, autophosphorylation takes place at a conserved Thr residue that stabilizes the active state of the enzyme. In this state, significant residual activity is still present after dissociation of Ca Vcalmodulin and the enzyme remains in an active state for a longer time after the Ca signal has died away. The active state is only terminated when the activating phosphate residue is cleaved off by a protein phosphatase.

Fig. 7.16. The dual function of protein kinases and protein phosphatases. Phosphorylation of proteins (PI, P2) can fix the latter into an active or inactive state. In the case of PI, protein kinases have an activating effect and protein phosphatases are inactivating the reverse is trne for P2.

Another mechanism of regulation of protein tyrosine phosphatases is via Ser/Thr phosphorylation. Specific phosphorylation of protein tyrosine phosphatases by Ser/ Thr-specific protein kinases of types A and C has been reported (see Neel and Tonks, 1997). This observation indicates the possibility that signal transductions via Ser/Thr kinases and via Tyr kinases/phosphatases may cooperate and that different signal pathways may be crosslinked in this way. 

In an effort to interfere with CML progression, pharmaceutical scientists at Novartis first cloned and produced recombinant B CR-AB L kinase. With the availability of this enzyme in sufficient quantity and purity, a mass in vitro screen of a series of enzyme inhibitors was implemented to identify drug candidates that produce an optimum pharmaceutical profile. From these, they identified STI571 (Gleevec) as a lead candidate to block the kinase activity of BCR-ABL. STI571 acts as a competitive inhibitor of ATP binding to the enzyme, which leads to the inhibition of tyrosine phosphorylation of proteins involved in BCR-ABL signal transduction [39,40]. 

The control of glycogen phosphorylase by the phosphorylation-dephosphorylation cycle was discovered in 1955 by Edmond Fischer and Edwin Krebs50 and was at first regarded as peculiar to glycogen breakdown. However, it is now abundantly clear that similar reactions control most aspects of metabolism.51 Phosphorylation of proteins is involved in control of carbohydrate, lipid, and amino acid metabolism in control of muscular contraction, regulation of photosynthesis in plants,52 transcription of genes,51 protein syntheses,53 and cell division and in mediating most effects of hormones. 

The MAPK cascade also has direct effects upon protein synthesis, i.e., on the translation of mRNA messages. For example, insulin stimulates phosphorylation of proteins that regulate a translation initiation factor, a protein called eIF-4E (see Chapter 29). Phosphorylation of inhibitory proteins allows them to dissociate from the initiation factor so that protein synthesis can proceed 485/486... 

Photosynthetic phosphorylation of protein side chains 79 substrate level 775, 800 Phosphorylation, photosynthetic. See Photosynthetic phosphorylation Phosphorylation reactions 303 Phosphorylation state ratio definition of 303 O-Phosphoserine 610s Phosphoserine 545 Phosphothreonine 545 Phosphotransferase system bacterial 419,420 Phosphotransferases 637... 

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