The Substrate Specificity of GSK3

Soon after its discovery, it was noted that GSK3 could only phosphorylate glycogen synthase efficiently if glycogen synthase had already been phosphorylated by CK2 6 . Phosphorylation by CK2 did not inhibit glycogen synthase, but primed this enzyme for phosphorylation by GSK3. Elegant studies by Roach and his colleagues then established that the substrate specificity requirements of GSK3 are unique the protein kinase phosphorylating serine and threo-nine residues that lie in Ser...

The Consensus Binding Site on Fc

Superposition of the binding site footprints of the four natural IgG-Fc binding domains reveals the presence of a common surface patch on the Fc surface (Fig. 3a). Just six side chains are involved in forming this saddle-shaped consensus site between the 250's loop of the CH2 domain and the 430's loop of the CH3 domain. Together these side chains Figure 3 Superposition of the binding site footprints on IgG-Fc for the natural Fc binding proteins (a). Surface atoms are colored red, yellow, light...

Budding Yeast Cdc14 is Essential for Exit from Mitosis

Following their association with B-type cyclins, the activation of cyclin-dependent kinases (Cdk) triggers the onset of mitosis. At anaphase after sister chromatids have separated, mitotic Cdks must be inactivated in order for cells to exit from mitosis. During exit from mitosis, cells restore the nucleus to its premitotic state (e.g., disassemble the mitotic spindle) and prepare for cytokinesis (for review, see Morgan 5 ). A prevailing mechanism for mitotic Cdk inactivation is the regulated...

Initiation of BCR Signaling Is Controlled by Redox Regulation

How signals are initiated upon antigen binding to the BCR is not completely understood and is still a matter of controversy. It has been found that the three protein tyrosine kinases (PTKs) Syk, Lyn, and Btk, as well as the adaptor protein SLP-65 BLNK and PLC-y2, are required for an optimal calcium response following engagement of the BCR (Fig. 1A) 6 . In the absence of Syk and Btk, no calcium response is observed, but in the absence of the Src-family kinase Lyn the amplitude of the calcium...

The Structure of the B Cell Antigen Receptor

The BCR is composed of two protein modules, the membrane-bound immunoglobulin (mIg) molecule and the Ig-a Ig-P heterodimer, which mediate antigen binding and signaling, respectively (Fig. 1A) 2,3 . The proper assembly between the mIg molecule and the Ig-a Ig-P heterodimer in the membrane of the endoplasmic reticulum is a prerequisite for the transport of the BCR to the cell surface. All five major classes of mIg (mIgM, mIgD, mIgG, mIgA, and mIgE) are associated with the Ig-a Ig-P heterodimer,...

Catalytic Activities of the PPP Family Members

PPPs dephosphorylate phosphoserine and phosphothreo-nine residues in proteins in vivo and in vitro. Mammalian PP1, PP2A, and PP2C have also been shown to dephosphorylate histidine residues in proteins in vitro 14 . Mammalian PPPs expressed in Escherichia coli display properties that are slightly different from the native enzymes, including dependence or partial dependence on divalent metal ions such as Mn2+ or Mg2+ 15 . Bacterially expressed mammalian Ppplc also exhibits protein-phosphotyrosine...

Interaction Domains A Common Theme in Signaling

Interaction domains are essential in signaling from many different types of cell-surface receptors, as well as in cellular events such as the cell cycle, protein and vesicle trafficking, targeted protein degradation, DNA repair, and control of the cytoskeleton. Thus, the SH2 domain serves as a prototype for a growing family of protein-interaction modules (Table 1), some of which specifically recognize posttranslationally modified motifs, in a fashion akin to the selective binding of SH2 and PTB...

Chemokine Structure and Function

Although their discovery has been relatively recent (mostly within the past 15 years), chemokines and their receptors have rapidly become appreciated for their impact on health and disease. They are involved in a broad variety of natural biological processes, including development, inflammation, immunity, and angiogenesis. In addition, these sequences have been corrupted by pathogens to subvert the innate and adaptive immune responses. This review provides a brief introduction to chemokine...

Bidentate PTP Inhibitors

Although pTyr is essential for peptide protein substrate recognition, pTyr alone does not possess high affinity for PTPs 45 . This and the fact that the PTP active site (pTyr binding site) is highly conserved among various PTPs present a serious challenge for the development of potent and selective PTP inhibitors targeted primarily to the active site. The discovery of a second aryl phosphate-binding site in PTP1B (defined by residues Arg24, Arg254, Met258, Gly259, and Gln262), which is not...

IgSuperfold Structures and Assemblies

The Ig-superfold is characterized by a primary sequence motif that spans some 100 amino acids. In three dimensions, this sequence motif translates into a compact domain structure that comprised of two anti-parallel P-sheets packed face to face (Fig. 1). Although there is a defined topology and connectivity for the Ig-superfold, the number of P-strands is variable. To take account of this variability Ig-like domains have been classified into different sets, according to the number and...

Conclusion

The development of effective nanomolar-binding inhibitors of the influenza virus NA is one of the success stories of structure-based drug design. The active site is remarkably rigid (Fig. 4e), except for one conserved glutamic acid, Glu276, which is free to rotate 90 about 2. This creates an extensive hydrophobic pocket in a region normally occupied by the glycerol group of the natural substrate. The most successful inhibitors have exploited this pocket to provide molecules with greater...

Mechanisms of GPCR Desensitization and Endocytosis

Heterotrimeric G Proteins Mediated by Receptor Phosphorylation Extensive studies of certain GPCRs, such as rhodopsin (a light-activated GPCR) and P2-AR (a ligand-activated GPCR), established a highly conserved mechanism that regulates the functional activity of many GPCRs 3-5,9 . This mechanism involves the phosphorylation of receptors by a specific family of G-protein-coupled receptor kinases (GRKs) followed by the interaction of phosphorylated receptors with cytoplasmic accessory proteins...

Cell Signaling Tomorrow

As the humane genome and importantly the genomes of several other key research paradigms reach completion in terms of sequence, interpretation, and full annotation, it will be possible to know, in a general sense, the complete complement of proteins, involved in cell signaling. Of course, this signaling proteome will contain a vast number of variants arising from message splicing and posttranslational modification. Appreciating how all of these variants interact as a function of time in...

Eph Receptor Signaling Via Cytoplasmic Protein Tyrosine Kinases

Upon ligand binding, Eph receptors are thought to dimer-ize, perhaps oligomerize, and autophosphorylate specific tyrosine residues of the cytoplasmic part of the partner receptor. Somewhat unusual among RTKs, Eph kinase activity is autoinhibited by the unphosphorylated juxtamembrane Figure 1 Eph receptor structure and cytoplasmic interactions. The intracellular part of Eph receptors consists of a juxtamembrane region, tyrosine kinase domain, SAM domain (of unknown function), and a PDZ binding...

High Affinity Variant of hGH hGHv Reveals an Altered Mode for Receptor Homodimerization

Using phage display mutagenesis selections, a variant of hGH that binds > 100-fold more tightly to hGH-R at site 1 was produced 41,42 . This variant (hGHv) has 15 mutations localized in its site 1 binding site and is fully biologically active but is totally specific to hGH-R, losing its ability to bind to hPRL-R. (This is a clinically relevant, second-generation hGH used for treating acromegaly). The recent high-resolution X-ray analysis of the ternary complex of hGHv bound to two copies of...

IRSProtein Structure and Function

Alignment of the amino acid sequences of the IRS-proteins reveals important similarities and subtle differences (Fig. 1). IRS 1-4 and Chico contain an NH2-terminal pleckstrin homology (PH) domain adjacent to a PTB domain. The structures of the PH and PTB domains are remarkably similar 30 , and both facilitate recruitment of IRS-proteins to the activated insulin and IGF1 receptors deletion of the PH and PTB domain almost completely prevents phosphoryla-tion of the C-terminus. The PTB domain...

The Transition State

In the transition state, noncovalent bonds are in the process of being made and broken. At least one encounter complex can be found prior to the transition state, with additional intermediates occupying the energy landscape past the transition state. What is the molecular basis of the transition state for protein-protein interactions The bound state of two proteins is characterized by local specific interactions (e.g., van der Waals, electrostatic) between widely desolvated binding sites,...

PP1 Regulatory or Targeting Subunits

Protein phosphatase 1 enzymes acquire specificity of function by their association with targeting regulatory pro teins that direct the enzyme to distinct subcellular structures or compartments in proximity to physiological substrates, confer substrate specificity and or modulate enzyme activity. Over 40 PPl-associated proteins are currently known. Most interact with PPlc through multiple sites, a shared site recognizable in many of the subunits, and other sites unique to the individual...

The Structure of the Atypical Kinase Domain Reveals Similarity to Classical Protein Kinases and to Metabolic Enzymes

The kinase domain of ChaK forms a dimer as a consequence of a domain-swapping exchange of an N-terminal 27-residue dimerization segment 17 . The isolated ChaK kinase domain is also a dimer in solution, and the dimeric property of ChaK may be relevant to the biological function or regulation of the kinase, as TRP channels, like other voltage-gated ion channels, are likely to be tetrameric. However, EF2 kinase and MHCK are monomers and would require an alternative extended polypeptide sequence to...

INAD Organizes Signaling Complexes

Phototransduction in Drosophila is one of the fastest G-protein-coupled signaling pathways known, taking less than 20 ms from light activation to maximum response. This high speed of signaling is primarily due to the incorporation of signaling components into multi-protein complexes. Signaling complexes bring components into close proximity, promoting rapid interaction as well as ensuring the proper sub-cellular localization of components. The central organizer of these signaling complexes is...

Summary

The LRR receptors represent the largest group of cell surface receptors in Arabidopsis thaliana and probably in all plants. Although this review has focused on the LRR receptor protein kinases, another 30 genes in Arabidopsis encode LRR receptors with a short cytoplasmic domain 31 . Although the functions of only of few of the Arabidopsis LRR receptors have been established, it is clear that these proteins have diverse roles in coordinating cellular signaling in plants. CLV1 and HAE belong to...

Oxyanions as PTP Inhibitors

Inorganic phosphate is a hydrolytic product of the PTP reaction and serves as a reversible competitive PTP inhibitor K 5 mM 20 . Because of the similar physical and chemical properties, many oxyanions, including sulfate, arsenate, molybdate, and tungstate, can competitively inhibit PTPs to various degrees 10 M to millimolar levels . These oxyanions could inhibit PTPs by simply mimicking the tetrahedral geometry of the phosphate ion. The crystal structure of the PTP complexed with tungstate...

Domain and Subunit Structure of PPP Family Members

Many protein serine threonine phosphatases in the PPP family are high-molecular-weight complexes containing one or more regulatory subunits. A large number of different regulatory subunits bind in a mutually exclusive manner to the Ppp1c catalytic subunit 24,25 or the Ppp2 PP2A core complex of the catalytic subunit and a regulatory subunit 17 . These interactions allow a single PPP catalytic subunit to participate in many different cellular functions. Details of these PPP regulatory subunits...

ZAP70Syk PTKs

Tyrosine phosphorylation of the receptor-encoded ITAMs mediates the binding of the ZAP-70 and Syk PTKs to the TCR. This family of cytoplasmic PTKs is unique in that they encode tandem SH2 domains that mediate their interaction with the TCR-encoded ITAMs 39,40 . While studies in transformed T-cell lines initially demonstrated that the TCR-encoded ITAMs undergo tyrosine phosphorylation following TCR crosslinking, subsequent studies of nontransformed T cells indicate that the receptor already...

Covalent PTP Modifiers

Sulfhydryl Group Example

The PTPs employ covalent catalysis, utilizing the thiol group of the active-site Cys residue as the attacking nucle-ophile to form a thiophosphoryl enzyme intermediate E-P 6 . Substitutions of the Cys residue completely abrogate PTP activity. The nucleophilic cysteine is housed within the active-site architecture specifically designed to bind a negatively charged phosphoryl group. Consequently, the pKa for the sulfhydryl group of the active-site Cys is extremely low 5 7 . Thus, the PTPs are very sensitive to thiol-specific alkylating agents. For example, the PTPs can be irreversibly inactivated by iodoacetate, N-ethylmaleimide, and 5,5'-dithio-2-nitrobenzoic acid 7-9 . In addition, PTPs can also be inactivated by heavy metals including Zn2 , Cu2 , and p- hydroxymercuri benzoate, possibly through covalent bond formation with the active-site thiol group. There were Figure 1 Structures of covalent inactivators of PTP. Figure 1 Structures of covalent inactivators of PTP. several attempts...

Regulation by Dimerization

Dimerization plays a critical role in the regulation of another family of transmembrane proteins, the receptor tyro-sine kinases. Specifically, ligand binding to the extracellular domain allows the intracellular kinase domain to dimerize and cross-phosphorylate at regulatory sites, leading to activation of the intracellular kinase domain. Two independent crystal structures of the membrane-proximal phosphatase domain, D1, of RPTPa reveal a symmetrical dimer in which the active site of one domain...

Erica Dutil Sonnenburg 2Tony Hunter and Joseph P Noel

Structural Biology Laboratory and 2Molecular and Cell Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California The receptor protein tyrosine phosphatases RPTPs are a family of transmembrane phosphatases that catalyze the removal of the phosphate moiety from a phosphotyrosine residue pTyr , resulting in a variety of intracellular responses including long-term potentiation, axonal path finding and neural transmission, and transformation. RPTPs consist of an...

Fabiola Janiak Spens and Ann H West

Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma Autophosphorylating histidine protein kinases are predominantly found in bacterial organisms and to a more limited extent in archaebacteria and nonvertebrate eukaryotic organisms where they function in two-component signal transduction pathways. These signaling systems typically consist of a sensor histidine kinase HK and a response regulator RR Fig. 1A . HKs are responsive to extracellular stimuli and can...

Comparative Kinomics

A great deal can be learned about the evolution of the protein kinase superfamily through the comparison of the kinomes of different eukaryotes 7,8 Figures 1 and 2 . Fifty one protein kinase families are common to all eukary-otes these serve cell essential functions examples are Cdk, CAMK, PKA, MAP kinase, etc. Interestingly, most of the atypical protein kinase families exist in all four kinomes yeasts, but clearly were not selected for diversification during evolution. Seven families are...

S4 Is the Primary Voltage Sensor

Electrical Voltage Ion Channels

A high-resolution structure of the voltage-sensing domain of an ion channel has yet to be obtained however, some things are already known about the structure of this domain. S1 to S6 form transmembrane helices 8,13,14,19,25,33 . The S4 helix is conserved across voltage-gated cation channels and contains a positively charged arginine R or lysine K at every third position Fig. 4 . Because S4 spans the membrane, some of these charges sense the electric field. The spacing of positive charges...

Introduction

Bovine Rhodopsin

G-protein-coupled receptors GPCRs represent a very large superfamily of receptors that are critical for signaling of a diverse group of ligands to heterotrimeric G proteins 1 . At least 30 of predicted potential drug targets are GPCRs 2 . Ligands for these receptors include light, odorants, tastes, small-molecule neurotransmitters, peptides, glycoprotein hormones, proteases, and others. GPCRs are composed of seven transmembrane segments, with an extracellular amino terminus and a cytoplasmic...

The Phosphatases

Cytokine signaling involves a cascade of tyrosine phosphorylation events. Tyrosine phosphorylation of many proteins is rapid but transient. It is therefore not surprising that dephosphorylation of activated signaling molecules by specific phosphatases is an important mechanism for the termination of cytokine signaling 4 . The Src homology domain 2 SH2 -containing tyrosine phosphatase SHP-1 has been identified as a critical negative regulator of cytokine signaling 5 . SHP-1 is a cytosolic...

Elizabeth Hewat

Institut de Biologie Structurale J-P Ebel, Grenoble, France The attachment of a virus to specific cell-surface receptors is a key event in the life cycle of animal viruses. It determines the host range and tropism of infection and initiates delivery of the genome into the cell. Once bound to a receptor, the non-enveloped viruses such as the rhinoviruses must then transfer their genome directly across a membrane into the cytoplasm for reproduction 1 . Human rhinoviruses HRVs are a major cause of...

Cytokine Signaling Proteins

Cytokines are a loosely defined set of secreted factors that control a variety of important biological responses related to hematopoiesis and immune function. There are over 40 members of this family of small 15-30 kDa glycoproteins, and many of them display functional redundancy 2 . Cytokines bind receptors that are characterized by sequence and structural similarities in their extracellular regions and the lack of catalytic domains in their intracellu-lar portions 3 . The intracellular...

PIAS Protein Inhibitors of Activated STATS

The PIAS family is composed of five members PIAS1, PIAS3, PIASxa, PIASxP, and PIASy 34 . PIAS proteins are greater than 50 homologous and contain several highly conserved regions, including a putative zinc binding motif and a highly acidic region. PIAS1 and PIAS 3 have been shown to negatively regulate cytokine-activated STAT-1 and STAT-3, respectively 34,35 . The specificity of their action has been demonstrated by in vitro studies PIAS1 co-immunoprecipiated with STAT-1 but not STAT-2 or...

Chao Zhang and 1 2Kevan M Shokat

[gama 32p Benzyl Atp

Department of Cellular amp Molecular Pharmacology, University of California, San Francisco, California 2Department of Chemistry, University of California, Berkeley, California A major challenge of the post-genomic era is to derive a complete map of all signaling networks in mammalian cells. One difficulty in mapping signaling pathways is determination of the substrates of each protein kinase 1,2 . Kinase substrates are difficult to identify chiefly because of the large size of the kinase...

LRR Receptor Protein Kinases The Functional View

Lrr Receptor

Although some information has been obtained from expression studies and biochemical analysis of the RLKs 1 , genetic approaches have provided the most information about the roles of the LRR RLKs in cellular communication. Of particular significance for this review is the fact that the functions of only five Arabidopsis RLKs have been described, all of which encode LRR RLKs. These Arabidopsis RLKs of known function include ERECTA ER , CLAVATA 1 CLV1 , HAESA HAE , BRASSINOSTEROID-INSENSITIVE 1...

Regulation of the Stress Activated MAP Kinase Cascades

One of the well-defined roles of PP2C in cell signaling is the downregulation of the stress-activated MAP kinase cascades in eukaryotes. A MAPK cascade is composed of three kinases mitogen-activated protein kinase MAPK , MAPK kinase MAPKK , and MAPKK kinase MAPKKK . Extracellular stimuli are transmitted into the nucleus through sequential phosphorylation of these kinases a signal-stimulated MAPKKK phosphorylates a specific MAPKK, which in turn phosphorylates the conserved threonine and tyrosine...

Multisite Phosphorylation Ubiquitination and Switch Like Responses

Photos Com

An important issue in the design of cell signaling networks is how protein-protein interactions can be used to integrate signals and create all-or-none responses. A typical Figure 2 A The Grb2 SH3-SH2-SH3 adaptor couples a pTyr-X-Asn docking site to multiple downstream targets through a series of protein-pro tein and protein-phospholipid interactions. One core pathway to cell growth is assembled through the N-terminal SH3 domain of Grb2 interacting with Pro-X-X-Pro motifs on the...

SOCS Suppressors of Cytokine Signaling Family

As with many breakthroughs, three groups simultaneously discovered suppressor of cytokine signaling 1 SOCS-1 , also known as JAK binding protein JAB and STAT-inducible STAT inhibitor 1 SSI-1 , using three very different strategies 1 SOCS-1 was cloned based on its ability to inhibit interleukin-6 IL-6 -induced macrophage differentiation of the monocytic cell line, M1 44 2 JAB was isolated in a yeast two-hybrid screen for JAK-2 binding proteins 45 3 SSI-1 was isolated by an antibody screen for...

Box 1 Modeling Ultrasensitivity with Stimulus Response Curves

The shape of a given systems stimulus-response curve is a key aspect of the steady-state behavior of a signaling system. Typical Michaelis-Menten enzymes exhibit hyperbolic stimulus response curves Fig. 3, nH 1 . At very low stimulus levels, the response grows linearly with the stimulus. As the extent of the stimulus increases, the response to each quanta of stimulus becomes progressively smaller. In other words, a Michaelian also referred to as graded or hyperbolic system obeys the law of...

Subcellular Localization of ErbB Proteins

Egfr Localization

The localization of LET-23 at the basolateral face of precursor epithelial cells is important for vulval development in C. elegans. In mammalian skeletal muscle, the clustering of ErbB2, ErbB3, and ErbB4 at the postsynaptic membrane of neuromuscular junctions is required for efficient binding to neuron-derived neuregulins and subsequent induction of acetylcholine receptor synthesis. In polarized epithelial cells, most EGFR and ErbB2 molecules are localized to the basolateral face where...

Conserved and Functionally Important Molecular Interactions

Although the Fc hinge region binding proteins are unrelated at the secondary and tertiary structural levels, patterns do emerge when one compares the detailed atomic interactions made by these molecules. Superposition of the available crystal structures enables identification of a set of conserved molecular interaction sites in the consensus region Fig. 4 . Although the overall folds of each of these Fc binding domains are distinct, there are numerous similarities in the geometric arrangements...

PTK Subfamilies

In the human genome, 58 genes encode PTK receptors 2,36 . Each receptor consists of an extracellular ligand-binding part, a single transmembrane domain, and an intra-cellular part with an intrinsic kinase domain. Based on their overall structures, the PTK receptors can be placed into 20 subfamilies Fig. 1 . Individual subfamilies are characterized by specific structural motifs in their extracellular parts e.g., Ig-like domains and fibronectin type III domains . Moreover, the sequences of the...

JAK Structure and Localization

Jak Kinase

The JAK kinases are relatively large proteins, with molecular weights of approximately 120 to 140 kDa. Seven regions Figure 2 Cytokine receptor families and associated JAKs. From left to right, 1 the homodimeric hormone receptors, which signal via JAK2 2 the IL-3 family of receptors, each having a unique ligand binding a chain and sharing a common beta Pc subunit associated with JAK2 3 the IL-6 subfamily of receptors, which have a common gp130 subunit that can interact with different members of...

IgGReceptor Interactions

Neonatal Receptor

Several crystal structures are now available for the extracellular domains of FcyRII 7-9 and FcyRIII 10,11 , which show together with FceRI 12,13 that they are all remarkably similar, as expected from their highly homologous sequences. There is an acute angle between the two Ig-like domains, which pack against each other around a hydrophobic interface. In the structure of the complex between FcyRIII and IgG Fc, first determined by Sondermann et al. in 2000 10 , loops from the a2 domain and part...

Pantophobiac After

Prior to the cloning of Ca2 -activated K channels, Kung and colleagues 43 studied behavioral mutants in Paramecium that exhibited an exaggerated response to noxious stimuli. Wild-type animals use the Ca2 that enters during a Ca2 -based action potential to activate two Ca2 -dependent conductances an inward Na and an outward K conductance that reverse ciliary motion, allowing the animal to move away from the noxious stimulus. Overreacting mutants, called pantophobiacs, lack the K conductance,...