Protein kinase specificity

Cytoplasmic serine/threonine protein kinases catalyze the transfer of phosphate groups to serine and threonine residues of target proteins. Serine/threonine kinases have been recognized as the products of protooncogenes (e.g., c-mos, c-raj) or as kinases intimately involved with the regulation of serine/threonine kinase activity by cAMP. Some of these kinases specifically phosphorylate cellular structural proteins, such as histone, laminins, etc. Others phosphorylate still more kinases, resulting in either the activation or deactivation of downstream protein kinases. Specific examples in which serine/threonine kinases elicit specific cellular responses are discussed in this chapter. 

How do a wide variety of neurotransmitters and hormones produce tissue- and cell-specific biological responses, if many such responses are mediated by the same intracellular messengers, cAMP and cAMP-depen-dent protein kinase Specificity is achieved at two levels at the level of tissue-specific receptors for the neurotransmitter or hormone and at the level of tissue-specific substrate proteins for the protein kinase. Only tissues that possess specific receptors will respond to a certain neurotransmitter or hormone. Moreover, since all cells contain very similar catalytic subunits of protein kinase A (see Ch. 23), the nature of the proteins that are phosphor-ylated in a given tissue depends on the types and amounts of proteins expressed in that tissue and on their accessibility to the protein kinase. 

Fig. 10.1. Principle of signal transduction through intracellular protein kinase cascades. The intracellular protein kinase cascades are organized in modules composed in most cases of three proteinkinases and a scaffold protein. The modules process signals that are registered, integrated and passed on at the inner side of the cell membrane by central switching stations such as the Ras protein or the Rac protein. In the case of the MAP kinase pathway, the cascade includes at least three different protein kinases. Specific regulatory processes may take effect at every level of the cascade in addition, signals may be passed from the different protein kinases to other signaling pathways.

Schaeffer, H.J. Weber, M.J. (1999) Mitogen-activated protein kinases specific messages from ubiquitous messengers. Mol. Cell. Biol. 19, 2435-2444. 

The regulation of translation through the phosphorylation of eIF-2 is best understood as it operates in the rabbit reticulocyte. Two protein kinases specific for the a subunit of eIF-2 have been purified from reticulocytes. One of these kinases, termed the heme-regulated inhibitor repressor (HRI), serves to coordinate the rate of hemoglobin synthesis (more than 90% of the total protein synthesized in the reticulocyte is hemoglobin) with the availability of hemin (the... 

Wong YFI, Lee TY, Liang HK et al. KinasePhos 2.0 a web server for identifying protein kinase-specific phosphorylation sites based on sequences and coupling patterns. Nucleic Acids Res 2007 35(Web Server issue) W588-594. 

Huang HD, Lee TY, Tzcng SW ct aL KinasePKos a web tool for identifying protein kinase-specific phosphorylation sites. Nucleic Acids Res 2005 33(Wcb Server issue) W226-229. 

C. Kinase Assay Using a Mitogen-Activated Protein Kinase-Specific Substrate... 

Li, S. and Wattenberg, E.V, Differential activation of mitogen-activated protein kinases by palytoxin and ouabain, two ligands for the Na, K -ATPase, Toxicol. Appl. Pharmacol. 151, 377, 1998. Schaeffer, H.J. and Weber, M.J., Mitogen-activated protein kinases specific messages from ubiquitous messengers, Mol. Cell Biol. 19, 2435, 1999. 

Abe Y, Matsumoto S, Kito K, Ueda N. 2000. Cloning and expression of a novel MAPKK-like protein kinase, lymphokine-activated killer T-cell-originated protein kinase, specifically expressed in the testis and activated lymphoid cells. J Biol Chem 275(28) 21525-21531. 

Herget T, Freitag M, Morbitzer M, Kupfer R, Stamminger T, Marschall M (2004) Novel chemical class of pUL97 protein kinase-specific inhibitors with strong anticytomegaloviral activity. Antimicrob Agents Chemother 48 4154 162... 

In addition, vinpocetine selectively inhibits a specific calcium, calmodulin-dependent cycHc nucleotide phosphodiesterase (PDF) isozyme (16). As a result of this inhibition, cycHc guanosine 5 -monophosphate (GMP) levels increase. Relaxation of smooth muscle seems to be dependent on the activation of cychc GMP-dependent protein kinase (17), thus this property may account for the vasodilator activity of vinpocetine. A review of the pharmacology of vinpocetine is available (18). 

FIGURE 15.2 Enzymes regulated by covalent modification are called interconvertible enzymes. The enzymes protein kinase and protein phosphatase, in the example shown here) catalyzing the conversion of the interconvertible enzyme between its two forms are called converter enzymes. In this example, the free enzyme form is catalytically active, whereas the phosphoryl-enzyme form represents an inactive state. The —OH on the interconvertible enzyme represents an —OH group on a specific amino acid side chain in the protein (for example, a particular Ser residue) capable of accepting the phosphoryl group. 

Phosphorylation by cAMP-dependent protein kinases inactivates the reductase. This inactivation can be reversed by two specific phosphatases (Figure 25.33). 

AKAPs are a diverse family of about 75 scaffolding proteins. They are defined by the presence of a structurally conserved protein kinase A (PKA)-binding domain. AKAPs tether PKA and other signalling proteins to cellular compartments and thereby limit and integrate cellular signalling processes at specific sites. This compartmentalization of signalling by AKAPs contributes to the specificity of a cellular response to a given external stimulus (e.g. a particular hormone or neurotransmitter). 

CREB stands for cyclic-AMP response element (CRE) binding protein and is a transcription factor. When phosphorylated by cyclic AMP- and cyclic GMP-dependent Protein Kinases or other protein kinases it binds to gene promoters that contain a specific binding site. After binding, the respective transcription activity is modulated. 

Mitogen activated protein kinase (MARK) cascades are three kinase modules activated by phosphorylation. The three kinase modules are composed of a MAPK, a MAPKK, and a MAPKKK. There are multiple members of each component of the MAPK cascade that are conserved from yeast to human. Activation of selective MAPK modules by specific stimuli regulates cell functions such as gene expression, adhesion, migration, differ entiation, and apoptosis. 

Activation of Mi, M3, and M5 mAChRs does not only lead to the generation of IP3 followed by the mobilization of intracellular Ca2+, but also results in the stimulation of phospholipase A2, phospholipase D, and various tyrosine kinases. Similarly, M2 and M4 receptor activation does not only mediate the inhibition of adenylyl cyclase, but also induces other biochemical responses including augmentation of phospholipase A2 activity. Moreover, the stimulation of different mAChR subtypes is also linked to the activation of different classes of mitogen-activated protein kinases (MAP kinases), resulting in specific effects on gene expression and cell growth or differentiation. 

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