Principles of cell signaling - UT Southwestern

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39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 622 SUBSTRATE 1 (Protein SER) H OH PRODUCT 1 (Phosphorylated protein) H N C C N C N C C N C H O CH 2 H O H O CH 2 H O Protein kinases are two substrate enzymes O - P O - O + O - P O O SUBSTRATE (Mg 2+. 2 ATP) Mg 2+ O - P O O O - O - O - P O O O - P O O CH 2 (Mg 2+. Triphosphate) Ribose PROTEIN KINASE PRODUCT (Mg 2+. 2 ADP) Mg 2+ O - P O O CH 2 Adenine Adenine O (Mg 2+. Diphosphate) Rib FIGURE 14.29 Protein kinases transfer the -phosphoryl group from ATP to serine, threonine, or tyrosine residues in protein substrates. Protein phosphorylation is the most common form of regulatory posttranslational modification. It occurs in all organisms, and it is estimated that about one-third of proteins in animals are at some time phosphorylated. Phosphorylation can stimulate or inhibit the catalytic activity of an enzyme, the affinity with which a protein binds other molecules, its subcellular localization, its ability to be further covalently modified, or its stability. Single phosphorylations may cause 500-fold or greater changes in activity, and proteins are often phosphorylated on multiple residues in complex and interacting patterns. Most protein phosphorylation in eukaryotes, and essentially all in animals, is catalyzed by protein kinases; dephosphorylation is catalyzed by phosphoprotein phosphatases. Both classes of enzymes are controlled by diverse mechanisms. In addition, proteins are often phosphorylated by multiple protein kinases, resulting in the generation of a range of activity states. This complexity allows inputs from different signaling pathways to be integrated into the resulting activity of the target. In bacteria, plants, and fungi, an additional protein phosphorylating system known as twocomponent signaling is vital. The protein kinases involved in two-component signaling are unrelated to the eukaryotic protein kinase superfamily and phosphorylate aspartate residues rather than serine, threonine, or tyrosine. Protein kinases transfer a phosphoryl group from ATP to Ser, Thr, and Tyr residues of protein substrates to form chemically stable phosphate esters, as shown in FIGURE 14.29. In animals, the distribution of phosphate among these three amino acid residues is uneven: ~90%-95% is on Ser, 5%-8% on Thr, and less than 1% on Tyr residues. The human genome contains approximately 500 genes that encode protein kinases, and many protein kinase mRNAs undergo alternative splicing. This makes the protein kinase gene superfamily one of the largest functional gene groups. The number and diversity of these enzymes emphasize the great and varied uses of protein kinases to regulate cellular functions. Although some protein kinases have a limited tissue and/or developmental distribution, many are ubiquitously expressed. Protein kinases are grouped according to their residue specificity. Protein kinases that phosphorylate Ser will usually also recognize Thr, hence the name protein Ser/Thr kinase. Multicellular organisms have protein Tyr kinases, which only recognize Tyr. Dual specificity protein kinases can phosphorylate Ser, Thr, and Tyr in the appropriately restricted substrate conformational context and are generally the most selective of the protein kinases. The analysis of the kinomes of several organisms has led to a more elaborate grouping derived from sequence relationships, shown in FIGURE 14.30, that also reflects to some extent on regulatory mechanisms and substrate specificity. For example, the AGC group is named for its founding members, cAMP-dependent protein kinase (PKA), cyclic GMP-dependent protein kinase (PKG), Ca2+, and phospholipiddependent protein kinase (PKC). These protein kinases are regulated by second messengers and prefer substrates that contain basic residues near the phosphorylation site. In addition to substrate specificity for amino acid residues, most protein kinases are also selective for local sequence surrounding the substrate site. Screening strategies have resulted in methods to predict if proteins contain consensus substrate sites for a wide variety of protein kinases. Antibodies can be used to identify and roughly quantitate protein phosphorylation at specific sites in proteins. Beyond local recognition, protein kinases may display marked substrate selectivity among similar proteins based on overall 622 CHAPTER 14 Principles of cell signaling
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 623 Human kinome tree FIGURE 14.30 The protein kinases in the human genome can be grouped according to sequence relationships that reveal seven major branches. The tyrosine kinases are contained within one major branch. The others are Ser/Thrspecific or dual specificity, and are named for the best described members: AGC from PKA, PKG, and PKC; CAMK from the calcium, calmodulin-dependent kinases; CMGC from CDKs, MAPKs, GSK3, Clks; CK1 from casein kinase 1; STE from Ste20, Ste11, and Ste7, the MAP4K, MAP3K, and MAP2K in the yeast mating pathway; and TKL, the Tyr kinase-like enzymes. Reproduced with permission from G. Manning, et al. 2002. Science. 298: 1912-1934. © 2002 AAAS. Photo courtesy of Gerard Manning, Salk Institute, and reprinted with permission of Cell Signaling Technology, Inc. (www.cellsignal.com). three-dimensional structure, for example, or among proteins that have been differentially covalently modified by phosphorylation or ubiquitination. In animal cells, some protein kinases are hormone receptors that span the plasma membrane. Some protein kinase receptors are protein serine/threonine kinases, such as the transforming growth factor- (TGF-)receptor, but the majority are protein tyrosine kinases, including receptors for insulin, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and other regulators of cell growth and differentiation. Other protein kinases are intrinsically soluble intracellular enzymes, although they may bind to one or more organellar membranes. X-ray crystallographic structures of protein kinases have revealed a wealth of information about their mechanism of activation. The conserved minimum catalytic core of a protein kinase contains about 270 amino acids, yielding a minimum molecular mass of about 30,000 Da. Within this core, there are two folded domains that form the active site at their interface, as shown in FIGURE 14.31. One or both of the conserved lysine (Lys) or aspartate (Asp) residues that are required for phosphoryl transfer are frequently mutated to disrupt kinase activity. A sequence near the active site, referred to as the activation loop, often undergoes a conformational rearrangement to generate active forms of the protein kinases and is the most common site of regulatory phosphorylation in 14.24 Protein phosphorylation/dephosphorylation is a major regulatory mechanism in the cell 623
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Principles of cell signaling - UT Southwestern

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