Protein Hardware for Signaling

Published on 01/03/2015 by admin

Filed under Basic Science

Last modified 01/03/2015

Print this page

rate 1 star rate 2 star rate 3 star rate 4 star rate 5 star
Your rating: none, Average: 0 (0 votes)

This article have been viewed 1770 times

CHAPTER 25 Protein Hardware for Signaling

This chapter introduces proteins that transduce signals in the cytoplasm: protein kinases, protein phosphatases, guanosine triphosphatases (GTPases), and adapter proteins. Remarkably, kinases and GTPases use the same strategy to operate molecular switches that carry information through signaling pathways: the simple addition and removal of inorganic phosphate. Protein kinases add phosphate groups to specific protein targets, and phosphatases remove them. GTPases bind guanosine triphosphate (GTP) and hydrolyze it to guanosine diphosphate (GDP) and inorganic phosphate, which dissociates. In both cases, the presence or absence of a single phosphate group switches a protein between active and inactive conformations.

Because addition of phosphate is reversible, both types of switches can be used as molecular timers that cycle on and off at tempos determined by the intrinsic properties of the switch and its environment. GTPases are active with bound GTP and switch off when they hydrolyze GTP to GDP. Similarly, phosphorylation activates many proteins but can inhibit others. In all of these examples, a single protein acts as a simple binary switch.

These molecular switches are often linked in series to form a signaling cascade that can both transmit and refine signals. Enzymes along signaling pathways (including kinases) often act as amplifiers. Turning on the binary switch of one enzyme molecule can produce many product molecules, each of which, in turn, may continue to propagate and amplify the original signal by activating downstream molecules. Other pathways involve negative feedback loops. Few signaling pathways are linear; instead, most branch and intersect, allowing cells to integrate information from multiple receptors and to control multiple effector systems simultaneously. Chapter 27 illustrates the functions of molecular switches in several signaling pathways.

Protein Phosphorylation

Phosphorylation is the most common posttranslational modification of proteins and regulates the activity of one or more proteins along most signaling pathways. Among other things, phosphorylation controls metabolic enzymes, cell motility, membrane channels, assembly of the nucleus, and cell cycle progression. Sometimes, phosphorylation turns a process on; sometimes, it turns one off. In either case, both the addition of a phosphate by a protein kinase and its removal by a protein phosphatase are required to achieve regulation.

For historical and practical reasons, it has been easier to study protein kinases than protein phosphatases, so most research and accounts of regulation by phosphorylation emphasize kinases (witness the 361,167 PubMed hits for “protein kinase” compared with 127,267 for “protein phosphatase” in August 2006). Furthermore, many researchers assumed incorrectly that phosphatases are always active, leading to a lack of interest in their roles in signaling reactions. Readers should not forget that both directions are important on this two-way street.

In eukaryotes, more than 99% of protein phosphorylation occurs on serine and threonine residues, but phosphorylation of tyrosine residues regulates many processes in animals (Fig. 25-1). Bacteria and Archaea use histidine and aspartate phosphorylation for signaling (see Fig. 27-11), but these modifications are little known in eukaryotes. Phosphohistidine and phosphoaspartate are more difficult to assay than are other phosphorylated residues, so pathways using these phosphoamino acids might have escaped detection.

Effects of Phosphorylation on Protein Structure and Function

Despite its small size, phosphate is well suited to cause changes in the activity of proteins. The addition of a phosphate group with two negative charges to a single amino acid can change the conformation of a protein or alter interactions with other molecules, including the interaction of substrates with enzymes. A phosphate group can alter the activity of a protein in sever-al ways:

Direct interference. A phosphate group can directly block the binding site for a ligand. For example, phosphorylation inhibits the metabolic enzyme isocitrate dehydrogenase by blocking substrate binding to the active site (Fig. 25-2). Both direct steric hindrance and electrostatic repulsion between the negatively charged phosphate and negatively charged substrates prevent substrate binding. Phosphorylation also can directly block protein assembly reactions, such as the polymerization of intermediate filaments (see Fig. 35-4) and binding of ADF/cofilin proteins to actin monomers and filaments (see Fig. 44-6).

Figure 25-3 protein kinase structures. A, Ribbon diagram and space-filling model of cAMP-dependent protein kinase with a nonhydrolyzable ATP analog (red) bound to the active site. The adenine base of the ATP fits into a hydrophobic cleft formed by b-sheets lining the interface of the two lobes. The phosphates bind to conserved residues in loops connecting the b-strands. (PDB file: 1CPK.) B, Space-filling model of PKA with bound inhibitory peptide PKI. The location of this inhibitory peptide revealed the binding site for protein substrates. (PDB file: 1FMO.) C, Ribbon diagram of c-Src. When tyrosine-527 is phosphorylated, the SH2 domain binds intramolecularly to the C-terminus, locking the kinase in an inactive conformation. The N-terminal SH3 domain binds intramolecularly to a proline-rich sequence (PPII helix) connecting the SH2 and kinase domains. NT and CT are the N- and C-terminal lobes of the kinase domain. D, Ribbon diagram of PKA bound to the RIa regulatory subunit. The pseudo substrate peptide (yellow) sits in the active site. Binding of cAMP to two sites on the RIa subunit causes a conformational change that dissociates RIa from the catalytic subunit. (PDB file: 1U7E.) E, Insulin receptor tyrosine kinase. Ribbon diagram and space-filling model with the catalytic loop in orange and the activation loop in green. (PDB file: 1IRK.) F, Space-filling model of insulin receptor tyrosine kinase triphosphorylated on the activation loop. This rearranges the activation loop, allowing substrates (pink with a white tyrosine side chain) access to the active site. AMP-PNP is a nonhydrolyzable analog of ATP with nitrogen bridging the b- and γ-phosphates. (PDB file: 1IR3.)

(E–F, Space-filling models courtesy of Steven Hubbard, New York University, New York.)

Protein Kinases

Protein kinases catalyze the transfer of the g-phosphate from adenosine triphosphate (or, rarely, guanosine triphosphate) to amino acid side chains of proteins. Protein kinases are important, as is evident from the remarkable number of genes: 116 in budding yeast (second only to transcription factor genes), 409 in nematode worms (second only to seven-helix receptor genes), and 518 in humans. Most protein kinases in eukaryotes are either serine/threonine kinases or tyrosine kinases (Appendix 25-1). The difference is that most serine/threonine kinases phosphorylate either serine and threonine but not tyrosine, while most tyrosine kinases phosphorylate tyrosine but not serine or threonine.

Most serine/threonine and tyrosine kinases had a common evolutionary origin and share similar structures and catalytic mechanisms, despite differences in substrate specificity. Tyrosine kinases emerged in animals after their divergence from fungi. Nevertheless, fungi have phosphotyrosine owing to two families of serine/threonine kinases that also phosphorylate tyrosine and three protein tyrosine phosphatases to reverse these reactions. A family of 40 “atypical” protein kinases had a separate origin from the major family. Lipid kinases have a catalytic domain related to typical protein kinases. They phosphorylate inositol phospholipids (see Fig. 26-7) or a few proteins.

The catalytic domain of eukaryotic protein kinases consists of about 260 residues in two lobes surrounding the ATP-binding pocket (Fig. 25-3). Despite extensive sequence divergence, all of these kinases have a similar polypeptide fold with conserved residues at critical positions required for catalysis.

Each kinase has a restricted range of protein substrates, so activation of a particular protein kinase changes the phosphorylation and activity of a discrete set of target proteins. Substrate specificity is achieved by selective binding of substrates to a groove on the surface of the kinase (Fig. 25-3B). This groove recognizes amino acids that flank the phosphorylatable residue and position the acceptor amino acid side chain in the active site. Typically, all substrates that bind a particular kinase have similar residues surrounding the target serine, threonine, or tyrosine (a consensus target sequence). For example, the consensus sequence for protein kinase A (PKA) is Arg-Arg-Gly-Ser/Thr-Ile. The arginines and isoleucine flanking the target serine or threonine residue specify binding to PKA. Interactions outside the catalytic site may also contribute to specific binding.

In addition to the catalytic domain, most protein kinases have other domains for regulation or localization (Fig. 25-4). Adapter domains, such as SH2, SH3, and pleckstrin homology domains (Fig. 25-11), target kinases to specific sites in the cell. Such localization can either bring together a kinase and its substrates or limit their interaction. Transmembrane segments anchor receptor kinases to membranes. Receptor tyrosine kinases usually have additional residues inserted in the kinase domain and at the C-terminus. Phosphorylation of tyrosines in these inserts creates binding sites for effector proteins with SH2 domains (see Figs. 27-6, 27-7, and 27-8).


Figure 25-4 protein kinase domain architecture. See Appendix 25-1 for definitions of the kinase names. Btk, Bruton tyrosine kinase; Ca, calcium-binding site; cAMP and cGMP, cyclic nucleotide–binding sites; CM, overlapping pseudosubstrate/calmodulin-binding site; DAG, diacylglycerol-binding site; FGFR, fibroblast growth factor receptor; Ig, immunoglobulin domains; PH, pleckstrin homology domain; PS, pseudosubstrate sequences; SH2 and SH3, Src-homology domains; TM, transmembrane domain.

Prokaryotes generally lack serine/threonine/tyrosine kinases but use a large family of histidine kinases for signal transduction (see Fig. 27-11). These prokaryotic kinases differ in structure, mechanism and evolution-ary origin from eukaryotic kinases. A few bacteria have acquired eukaryotic kinases by lateral transfer of genes.

Regulation of Protein Kinases

Each kinase has its own regulatory mechanism, but most involve one or more of three strategies: (1) phosphorylation, (2) interactions with intrinsic peptides or extrinsic subunits that may themselves be targets for second messengers or regulatory proteins, and (3) targeting to specific cellular locations, such as the nucleus, plasma membrane, or cytoskeleton, enhancing interaction with specific substrates.


Phosphorylation can either activate or inhibit protein kinases. In some cases, another kinase molecule of the same type carries out the phosphorylation, but often another type of kinase is responsible. When linked in series, different types of kinases form signaling cascades that can amplify and sharpen the response to a stimulus (see Fig. 27-5):

Regulation of Substrate Binding

Peptides that are intrinsic to the kinase or part of a separate protein can inhibit kinases by competing with protein substrates for binding to the enzyme (Figs. 25-3B and D and 25-4):

Extrinsic regulation by inhibitory subunits. Separate regulatory (R) subunits inhibit PKA by blocking the protein substrate site with a pseudosubstrate (Figs. 25-3D and 25-4). Pseudosubstrates have consensus target sequences lacking the phosphorylated residue. For example, RI pseudosubstrate has the sequence Arg-Arg-Gly-Ala-Ile, which binds in the substrate groove but is not phosphorylated, as it has alanine or glycine, rather than serine, at the phosphorylation site. The RII pseudosubstrate has a serine, which is phosphorylated but then does not dissociate from the catalytic subunit as phosphorylated substrates do. Cyclic adenosine monophosphate (cAMP) regulates the affinity of these regulatory subunits for the catalytic subunit. In resting cells, the regulatory subunit is free of cAMP and binds the catalytic subunit with high affinity. With a rise in cAMP concentration (see Fig. 26-3), cAMP binds the regulatory subunit, dissociates it from the catalytic subunit, and allows substrates access to the active site.
Autoinhibition. Many kinases have an intrinsic pseudosubstrate sequence (see Fig. 25-4) that binds intramolecularly to the active site, autoinhibiting the enzyme (Fig. 25-3B). Ca2+-calmodulin activates myosin light-chain kinase and calmodulin-activated kinase (CaMK) by binding immediately adjacent to the pseudosubstrate and displacing the inhibitory peptide from the kinase. Cyclic guanosine monophosphate (cGMP) binding to protein kinase G (PKG) displaces the autoinhibitory peptide from the catalytic domain, activating the enzyme.


Several mechanisms target kinases to specific cellular locations, bringing them close to particular substrates. This targeting helps to explain how kinases with broad specificity can have specific effects in particular target cells:

Pleckstrin homology (PH) domains (Fig. 25-10A) and lipid tags target some kinases to lipid bilayers. A PH domain directs PKB/Akt to membrane polyphosphoinositides. This interaction with lipids opens up sites on the catalytic domain for phosphorylation and activation by PDK1, another kinase with a pleckstrin homology domain. An N-terminal myristic acid anchors Src tyrosine kinase to the plasma membrane.

Protein Phosphatases

Eukaryotes have several families of protein phosphatases that remove phosphate from amino acid side chains (Table 25-1 and Fig. 25-5). Like protein kinases, most protein phosphatases are active toward either phosphoserine/threonine or phosphotyrosine, although several dual-specificity phosphatases can dephosphorylate all three residues. The 90 active protein tyrosine phosphatases far outnumber the 20 serine/threonine phosphatase genes in the human genome. Each tyrosine phosphatase is thought to act on a limited number of substrates. The small number of serine/threonine phosphatases achieve specificity by associating with an array of accessory subunits, which regulate enzyme activity and target catalytic subunits to particular substrates. Domains flanking the catalytic domains also regulate enzyme activity (Fig. 25-6).

PPP Family of Serine/Threonine Phosphates

Members of the PPP family of serine/threonine phosphatases are found in Bacteria, Archaea, and all tissues of eukaryotes. PP1 and PP2A are two of the most evolutionarily conserved enzymes. All three PPP subfamilies share the same catalytic fold with a two-metal ion cluster (Fe2+ and Zn2+ in vivo) in the active site (Fig. 25-5A). Diverse regulatory subunits restrict the substrates for PP1 and PP2A by targeting catalytic subunits to specific sites in the cell, as illustrated by the following examples:

PP1: More than 50 associated proteins target a 38-kD catalytic subunit of PP1 to specific substrates. For example, M subunits target PP1 to myosin-II, where dephosphorylation of regulatory light chains relaxes smooth muscle (see Fig. 39-21). The complex of PP1 with an M subunit creates an active site that is specific for myosin-II light chains relative to other substrates. G subunits regulate glucose metabolism by targeting PP1 to glycogen particles, where it dephosphorylates two enzymes that control glycogen metabolism. Dephosphorylation inactivates glycogen phosphorylase, turning off glycogen breakdown, and activates glycogen synthase. The hormone adrenaline stimulates cells to mobilize energy stores by breaking down glycogen (see Fig. 27-3

Buy Membership for Basic Science Category to continue reading. Learn more here