Protein phosphorylation serine

The serine residue of isocitrate dehydrogenase that is phos-phorylated by protein kinase lies within the active site of the enzyme. This situation contrasts with most other examples of covalent modification by protein phosphorylation, where the phosphorylation occurs at a site remote from the active site. What direct effect do you think such active-site phosphorylation might have on the catalytic activity of isocitrate dehydrogenase (See Barford, D., 1991. Molecular mechanisms for the control of enzymic activity by protein phosphorylation. Bioehimiea et Biophysiea Acta 1133 55-62.)... 

In addition, eNOS is subject to protein phosphorylation. It can be phosphotylated on several serine (Ser), threonine (Thr), and tyrosine (Tyr) residues however, major changes in enzyme function have been reported for the phosphorylation of amino acid residues Seri 177 and Thr495 (in the human eNOS sequence) (Fig. 3). 

Phosphorylation is the reversible process of introducing a phosphate group onto a protein. Phosphorylation occurs on the hydroxyamino acids serine and threonine or on tyrosine residues targeted by Ser/Thr kinases and tyrosine kinases respectively. Dephosphorylation is catalyzed by phosphatases. Phosphorylation is a key mechanism for rapid posttranslational modulation of protein function. It is widely exploited in cellular processes to control various aspects of cell signaling, cell proliferation, cell differentiation, cell survival, cell metabolism, cell motility, and gene transcription. 

Given that these proteins have properly assembled, the initiation complex is ready to start transcription. How does the enzyme get started A component of TFIID, again a multi-subunit complex TFIIH, unwinds the DNA and phosphorylates serine-5 of the C-terminal tail (CTD) of the largest polymerase subunit (Rpbl). Serine-5 phosphorylation and phosphorylation of serine-2 (by pTEFb) are required to release the enzyme from the other components of the initiation complex and to start RNA synthesis. 

Chromatin remodeling, transcription factor modification by various enzyme activities, and the communication between the nuclear receptors and the basal transcription apparatus are accomplished by protein-protein interactions with one or more of a class of coregulator molecules. The number of these coregulator molecules now exceeds 100, not counting species variations and splice variants. The first of these to be described was the CREB-binding protein, CBP. CBP, through an amino terminal domain, binds to phosphorylated serine 137 of CREB and mediates transactivation in response to cAMP. It thus is described as a coactivator. CBP and... 

FIGURE 1 2-2 Schematic diagram of the phosphorylation sites on each of the four 60kDa subunits of tyrosine hydroxylase (TOHase). Serine residues at the N-terminus of each of the four subunits of TOHase can be phosphorylated by at least five protein kinases. (J), Calcium/calmodulin-dependent protein kinase II (CaM KII) phosphorylates serine residue 19 and to a lesser extent serine 40. (2), cAMP-dependent protein kinase (PKA) phosphorylates serine residue 40. (3), Calcium/phosphatidylserine-activated protein kinase (PKC) phosphorylates serine 40. (4), Extracellular receptor-activated protein kinase (ERK) phosphorylates serine 31. (5), A cdc-like protein kinase phosphorylates serine 8. Phosphorylation on either serine 19 or 40 increases the activity of TOHase. Serine 19 phosphorylation requires the presence of an activator protein , also known as 14-3-3 protein, for the expression of increased activity. Phosphorylation of serines 8 and 31 has little effect on catalytic activity. The model shown includes the activation of ERK by an ERK kinase. The ERK kinase is activated by phosphorylation by PKC. (With permission from reference [72].)... 

Several key questions remain with regard to the regulation of tyrosine hydroxylase by phosphorylation. What is the precise effect of the phosphorylation of each of these serine residues on the catalytic activity of the enzyme How does the phosphorylation of multiple residues affect enzyme activity Does the phosphorylation of one residue affect the ability of the others to be phosphorylated Tyrosine hydroxylase provides a striking example as to how multiple intracellular messengers and protein kinases converge functionally through the phosphorylation of a single substrate protein. Phosphorylation of tyrosine hydroxylase by cAMP-dependent and Ca2+-dependent protein kinases and by MAPK cascades... 

For example, c-Fos is heavily phosphorylated on a series of serine residues in the C-terminal domain of the protein by several types of protein kinases. The likely functional importance of these phosphorylation sites is indicated by the fact that the difference between c-Fos (the normal cellular form of the protein) and v-Fos (the viral oncogene product) is a frameshift mutation in the v-Fos protein, which obliterates the phosphorylated serine residues. It is speculated that the loss of these phosphorylation sites removes one mechanism by which the cell can regulate the protein, thereby leading to cellular transformation. 

Protein phosphorylation is one of the most important mechanisms in the regulation of cellular function. Proteins can be phosphorylated on serine, threonine or tyrosine residues. Most phosphorylation occurs on serine and threonine, with less than 1% on tyrosine (see Ch. 23). This perhaps accounts for the late discovery of tyrosine phosphorylation, which was found first on polyoma virus middle T antigen in 1979 by Hunter and colleagues [1,2]. 

Other PTMs may involve changes in the chemical nature of amino acids (e.g., citrullination or deimination). Because many of these modifications result in mass changes that are measurable by MS, they are amenable to detection by MS-based approaches. A number of emerging MS-based strategies allow the identification of PTMs. Several MS-based methods to determine the types and sites of protein phosphorylation and ubiquitination have been developed. Phosphorylation occurs mainly on serine, threonine, and tyrosine residues at a frequency ratio of 1800 200 1 in vertebrates.70 Although the phosphorylation of tyrosine residues occurs less frequently in the proteome, it has been extensively studied. 

Only three amino acids have a hydroxyl functional group in their side chain tyrosine, serine and threonine. Some kinases target only tyrosine residues (tyrosine kinases) whereas others may phosphorylate serine or threonine (Ser/Thr kinases). An enzyme protein (the substrate for the kinase) may have several tyrosine, serine or threonine residues within its primary sequence, but only some of these are subject to phosphorylation by a particular kinase (see Figure 3.6)... 

Phosphorylation has also been implicated in viral growth. A recent study showed that a lithium-sensitive kinase (e.g., GSK-3/3) phosphorylated serine 54 of the human respiratory syncytial virus (HRSV) structural P protein, which is a cofactor of the viral RNA polymerase. " The removal of the phosphate is controlled by phosphatase 2A (PP2A). When lithium-sensitive kinases were inhibited, the virus was unable to replicate and did not survive (Figure 3). 

Action. cAMP is an allosteric effector of protein kinase A (PK-A, [3]). in the inactive state, PK-A is a heterotetramer (C2R2), the catalytic subunits of which (C) are blocked by regulatory units (R autoinhibition). When cAMP binds to the regulatory units, the C units separate from the R units and become enzymatically active. Active PK-A phosphorylates serine and threonine residues of more than 100 different proteins, enzymes, and transcription factors, in addition to cAMP, cCMP also acts as a second messenger, it is involved in sight (see p. 358) and in the signal transduction of NO (see p. 388). 

Kinase inhibitors such as H7, which inhibits serine threonine kinases, resulted in sustained JAK2/STAT activation, suggesting that besides protein dephosphorylation, protein phosphorylation is also required. Furthermore, both inhibition of protein synthesis with cycloheximide and inhibition of... 

NMR and kinetic studies have been conducted with the hope of providing more details about the position and conformation of the polypeptide substrate in cAMP-dependent protein kinase. These have served to narrow down the possible spatial relationships between enzyme bound ATP and the phosphorylated serine. Thus, a picture of the active site that is consistent with the available data can be drawn (12,13,66,67). Although these studies have been largely successful at eliminating some classes of secondary polypeptide structure such as oi-hellces, 6-sheets or an obligatory 6-turn conformation 66), the precise conformation of the substrate is still not known. The data are consistent with a preference for certain 6-turn structures directly Involving the phosphorylated serine residue. However, they are also consistent with a preference or requirement for either a coil structure or some nonspecific type of secondary structure. Models of the ternary active-site complexes based on both the coil and the, turn conformations of one alternate peptide substrate have" been constructed (12). These two models are consistent with the available kinetic and NMR data. 

It has been shown that phosphorylation changes the local conformation of a protein and thereby affects the activity of the complete protein. 341 Phosphorylation of serine and threonine side chains often occurs (Scheme 2). Phosphoamino acids are readily characterized using 3H and 31P NMR experiments. The H and 31P NMR parameters are distinct for phosphorylated serine, threonine, and tyrosine and have also been used to identify both cis-and trans-O-phospho-4-hydroxy-L-proline. 35 Phosphorylation of Cys is rare, but it can be identified by NMR even in large proteins. 36 ... 

Our results demonstrated that the identified subsets of the activated protein kinases significantly increased the accuracy of clinical outcome predictions. Most notably in the study, we evaluated protein phosphorylation levels instead of total protein expression levels. Protein phosphorylation and dephosphorylation are well-characterized biochemical processes for protein kinases to conduct cellular signal transduction. Phosphorylation at certain tyrosine, serine, or threonine residues in kinases is a key step for their activation, and the measurement of these phosphorylations reflects their functional status in vivo. Thus, the protein kinase phosphorylation-based tissue microarray more accurately reveals the molecular mechanisms of breast cancers, and more accurately predicts the individualized survival and treatment response. 

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