241 human active and 13 inactive phosphatases in total;
194 phosphatases have substrate data;
336 protein substrates;
83 non-protein substrates;
1215 dephosphorylation interactions;
299 KEGG pathways;
876 Reactome pathways;
last scientific update: 11 Mar, 2019
last maintenance update: 01 Sep, 2023
The mRNA surveillance pathway is a quality control mechanism that detects and degrades abnormal mRNAs. These pathways include nonsense-mediated mRNA decay (NMD), nonstop mRNA decay (NSD), and no-go decay (NGD). NMD is a mechanism that eliminates mRNAs containing premature translation-termination codons (PTCs). In vertebrates, PTCs trigger efficient NMD when located upstream of an exon junction complex (EJC). Upf3, together with Upf1 and Upf2, may signal the presence of the PTC to the 5'end of the transcript, resulting in decapping and rapid exonucleolytic digestion of the mRNA. In the NSD pathway, which targets mRNAs lacking termination codons, the ribosome is believed to translate through the 3' untranslated region and stall at the end of the poly(A) tail. NSD involves an eRF3-like protein, Ski7p, which is hypothesized to bind the empty A site of the ribosome and recruit the exosome to degrade the mRNA from the 3' end. NGD targets mRNAs with stalls in translation elongation for endonucleolytic cleavage in a process involving the Dom34 and Hbs1 proteins.
Sphingomyelin (SM) and its metabolic products are now known to have second messenger functions in a variety of cellular signaling pathways. Particularly, the sphingolipid metabolites, ceramide (Cer) and sphingosine-1-phosphate (S1P), have emerged as a new class of potent bioactive molecules. Ceramide can be generated de novo or by hydrolysis of membrane sphingomyelin by sphingomyelinase (SMase). Ceramide is subsequently metabolized by ceramidase to generate sphingosine (Sph) which in turn produces S1P through phosphorylation by sphingosine kinases 1 and 2 (SphK1, 2). Both ceramide and S1P regulate cellular responses to stress, with generally opposing effects. S1P functions as a growth and survival factor, acting as a ligand for a family of G protein-coupled receptors, whereas ceramide activates intrinsic and extrinsic apoptotic pathways through receptor-independent mechanisms.
During meiosis, a single round of DNA replication is followed by two rounds of chromosome segregation, called meiosis I and meiosis II. At meiosis I, homologous chromosomes recombine and then segregate to opposite poles, while the sister chromatids segregate from each other at meoisis II. In vertebrates, immature oocytes are arrested at the PI (prophase of meiosis I). The resumption of meiosis is stimulated by progesterone, which carries the oocyte through two consecutive M-phases (MI and MII) to a second arrest at MII. The key activity driving meiotic progression is the MPF (maturation-promoting factor), a heterodimer of CDC2 (cell division cycle 2 kinase) and cyclin B. In PI-arrested oocytes, MPF is initially inactive and is activated by the dual-specificity CDC25C phosphatase as the result of new synthesis of Mos induced by progesterone. MPF activation mediates the transition from the PI arrest to MI. The subsequent decrease in MPF levels, required to exit from MI into interkinesis, is induced by a negative feedback loop, where CDC2 brings about the activation of the APC (anaphase-promoting complex), which mediates destruction of cyclin B. Re-activation of MPF for MII requires re-accumulation of high levels of cyclin B as well as the inactivation of the APC by newly synthesized Emi2 and other components of the CSF (cytostatic factor), such as cyclin E or high levels of Mos. CSF antagonizes the ubiquitin ligase activity of the APC, preventing cyclin B destruction and meiotic exit until fertilization occurs. Fertilization triggers a transient increase in cytosolic free Ca2+, which leads to CSF inactivation and cyclin B destruction through the APC. Then eggs are released from MII into the first embryonic cell cycle.
Autophagy is a degradative pathway for the removal of cytoplasmic materials in eukaryotic cells, and is characterized by the formation of a double-membrane structure called the autophagosome, either in a housekeeping capacity or during stress and senescence. The process of autophagy could be divided into several stages: induction, vesicle nucleation, elongation and closure, and fusion and digestion. Most essential autophagic machineries are conserved throughout eukaryotes (see map04140 for animals and map04138 for fungi). This map is for other eukaryotes including plants and protists, where autophagy related genes (ATGs) play similar roles in the life cycle. However, autophagy has been relatively less studied in lower eukaryotes.
Autophagy (or macroautophagy) is a cellular catabolic pathway involving in protein degradation, organelle turnover, and non-selective breakdown of cytoplasmic components, which is evolutionarily conserved among eukaryotes and exquisitely regulated. This progress initiates with production of the autophagosome, a double-membrane intracellular structure of reticular origin that engulfs cytoplasmic contents and ultimately fuses with lysosomes for cargo degradation. Autophagy is regulated in response to extra- or intracellular stress and signals such as starvation, growth factor deprivation and ER stress. Constitutive level of autophagy plays an important role in cellular homeostasis and maintains quality control of essential cellular components.
The phosphatidylinositol 3' -kinase(PI3K)-Akt signaling pathway is activated by many types of cellular stimuli or toxic insults and regulates fundamental cellular functions such as transcription, translation, proliferation, growth, and survival. The binding of growth factors to their receptor tyrosine kinase (RTK) or G protein-coupled receptors (GPCR) stimulates class Ia and Ib PI3K isoforms, respectively. PI3K catalyzes the production of phosphatidylinositol-3,4,5-triphosphate (PIP3) at the cell membrane. PIP3 in turn serves as a second messenger that helps to activate Akt. Once active, Akt can control key cellular processes by phosphorylating substrates involved in apoptosis, protein synthesis, metabolism, and cell cycle.
AMP-activated protein kinase (AMPK) is a serine threonine kinase that is highly conserved through evolution. AMPK system acts as a sensor of cellular energy status. It is activated by increases in the cellular AMP:ATP ratio caused by metabolic stresses that either interfere with ATP production (eg, deprivation for glucose or oxygen) or that accelerate ATP consumption (eg, muscle contraction). Several upstream kinases, including liver kinase B1 (LKB1), calcium/calmodulin kinase kinase-beta (CaMKK beta), and TGF-beta-activated kinase-1 (TAK-1), can activate AMPK by phosphorylating a threonine residue on its catalytic alpha-subunit. Once activated, AMPK leads to a concomitant inhibition of energy-consuming biosynthetic pathways, such as protein, fatty acid and glycogen synthesis, and activation of ATP-producing catabolic pathways, such as fatty acid oxidation and glycolysis.
Cardiac myocytes express at least six subtypes of adrenergic receptor (AR) which include three subtypes of beta-AR (beta-1, beta-2, beta-3) and three subtypes of the alpha-1-AR (alpha-1A, alpha-1B, and alpha-1C). In the human heart the beta-1-AR is the pre- dominate receptor. Acute sympathetic stimulation of cardiac beta-1-ARs induces positive inotropic and chronotropic effects, the most effective mechanism to acutely increase output of the heart, by coupling to Gs, formation of cAMP by adenylyl cyclase (AC), and PKA- dependent phosphorylation of various target proteins (e.g., ryanodine receptor [RyR]; phospholamban [PLB], troponin I [TnI], and the L-type Ca2+ channel [LTCC]). Chronic beta-1-AR stimulation is detrimental and induces cardiomyocyte hypertrophy and apoptosis. beta-2-AR coupled to Gs exerts a proapoptotic action as well as beta-1-AR, while beta-2-AR coupled to Gi exerts an antiapoptotic action.
The transforming growth factor-beta (TGF-beta) family members, which include TGF-betas, activins and bone morphogenetic proteins (BMPs), are structurally related secreted cytokines found in species ranging from worms and insects to mammals. A wide spectrum of cellular functions such as proliferation, apoptosis, differentiation and migration are regulated by TGF-beta family members. TGF-beta family member binds to the Type II receptor and recruits Type I, whereby Type II receptor phosphorylates and activates Type I. The Type I receptor, in turn, phosphorylates receptor-activated Smads ( R-Smads: Smad1, Smad2, Smad3, Smad5, and Smad8). Once phosphorylated, R-Smads associate with the co-mediator Smad, Smad4, and the heteromeric complex then translocates into the nucleus. In the nucleus, Smad complexes activate specific genes through cooperative interactions with other DNA-binding and coactivator (or co-repressor) proteins.
Hippo signaling is an evolutionarily conserved signaling pathway that controls organ size from flies to humans. In humans and mice, the pathway consists of the MST1 and MST2 kinases, their cofactor Salvador and LATS1 and LATS2. In response to high cell densities, activated LATS1/2 phosphorylates the transcriptional coactivators YAP and TAZ, promoting its cytoplasmic localization, leading to cell apoptosis and restricting organ size overgrowth. When the Hippo pathway is inactivated at low cell density, YAP/TAZ translocates into the nucleus to bind to the transcription enhancer factor (TEAD/TEF) family of transcriptional factors to promote cell growth and proliferation. YAP/TAZ also interacts with other transcriptional factors or signaling molecules, by which Hippo pathway-mediated processes are interconnected with those of other key signaling cascades, such as those mediated by TGF-beta and Wnt growth factors.
Tight junctions (TJs) are essential for establishing a selectively permeable barrier to diffusion through the paracellular space between neighboring cells. TJs are composed of at least three types of transmembrane protein -occludin, claudin and junctional adhesion molecules (JAMs)- and a cytoplasmic 'plaque' consisting of many different proteins that form large complexes. These are proposed to be involved in junction assembly, barrier regulation, cell polarity, gene transcription, and other pathways.
Dopamine (DA) is an important and prototypical slow neurotransmitter in the mammalian brain, where it controls a variety of functions including locomotor activity, motivation and reward, learning and memory, and endocrine regulation. Once released from presynaptic axonal terminals, DA interacts with at least five receptor subtypes in the central nervous system (CNS), which have been divided into two groups: the D1-like receptors (D1Rs), comprising D1 and D5 receptors, both positively coupled to adenylyl cyclase and cAMP production, and the D2-like receptors (D2Rs), comprising D2, D3, and D4 receptors, whose activation results in inhibition of adenylyl cyclase and suppression of cAMP production. In addition, D1Rs and D2Rs modulate intracellular Ca2+ levels and a number of Ca2+ -dependent intracellular signaling processes. Through diverse cAMP- and Ca2+-dependent and - independent mechanisms, DA influences neuronal activity, synaptic plasticity, and behavior. Presynaptically localized D2Rs regulate synthesis and release of DA as the main autoreceptor of the dopaminergic system.
Cerebellar long-term depression (LTD), thought to be a molecular and cellular basis for cerebellar learning, is a process involving a decrease in the synaptic strength between parallel fiber (PF) and Purkinje cells (PCs) induced by the conjunctive activation of PFs and climbing fiber (CF). Multiple signal transduction pathways have been shown to be involved in this process. Activation of PFs terminating on spines in dendritic branchlets leads to glutamate release and activation of both AMPA and mGluRs. Activation of CFs, which make multiple synaptic contacts on proximal dendrites, also via AMPA receptors, opens voltage-gated calcium channels (VGCCs) and causes a generalized influx of calcium. These cellular signals, generated from two different synaptic origins, trigger a cascade of events culminating in a phosphorylation-dependent, long-term reduction in AMPA receptor sensitivity at the PF-PC synapse. This may take place either through receptor internalization and/or through receptor desensitization.
Trypanosoma cruzi is an intracellular protozoan parasite that causes Chagas disease. The parasite life cycle involves hematophagous reduviid bugs as vectors. Once parasites enter the host body, they invade diverse host cells including cardiomyocytes. Establishment of infection depends on various parasite molecules such as cruzipain, oligopeptidase B, and trans-sialidase that activate Ca2+ signaling. Internalized parasites escape from the parasitophorous vacuole using secreted pore-forming TcTOX molecule and replicate in the cytosol. Multiplied parasites eventually lyse infected host cells and are released in the circulation. During these events, the parasites manipulate host innate immunity and elicit cardiomyocyte hypertrophy. T lymphocyte responses are also disturbed.
Hepatitis C virus (HCV) is a major cause of chronic liver disease. The HCV employ several strategies to perturb host cell immunity. After invasion, HCV RNA genome functions directly as an mRNA in the cytoplasm of the host cell and forms membrane-associated replication complexes along with non-structural proteins. Viral RNA can trigger the RIG-I pathway and interferon production during this process. Translated HCV protein products regulate immune response to inhibit the action of interferon. HCV core and NS5A proteins appear to be the most important molecules with regulatory functions that modulate transcription, cellular proliferation, and apoptosis.
Human papillomavirus (HPV) is a non-enveloped, double-stranded DNA virus. HPV infect mucoal and cutaneous epithelium resulting in several types of pathologies, most notably, cervical cancer. All types of HPV share a common genomic structure and encode eight proteins: E1, E2, E4, E5, E6, and E7 (early) and L1 and L2 (late). It has been demonstrated that E1 and E2 are involved in viral transcription and replication. The functions of the E4 protein is not yet fully understood. E5, E6, and E7 act as oncoproteins. E5 inhibits the V-ATPase, prolonging EGFR signaling and thereby promoting cell proliferation. The expression of E6 and E7 not only inhibits the tumor suppressors p53 and Rb, but also alters additional signalling pathways. Among these pathways, PI3K/Akt signalling cascade plays a very important role in HPV-induced carcinogenesis. The L1 and L2 proteins form icosahedral capsids for progeny virion generation.
During S phase of the cell cycle, RB1 is dephosphorylated by the PP2A protein phosphatase complex. Unphosphorylated RB1 associates with DNA damage sites in S phase, preventing initiation of DNA replication from these sites (Knudsen et al. 2000, Avni et al. 2003)
Sprouty was initially characterized as a negative regulator of FGFR signaling in Drosophila. Human cells contain four genes encoding Sprouty proteins, of which Spry2 is the best studied and most widely expressed. Spry proteins modulate the duration and extent of signaling through the MAPK cascade after FGF stimulation, although the mechanism appears to depend on the particular biological context. Some studies have suggested that Sprouty binds to GRB2 and interferes with the recruitment of GRB2-SOS1 to the receptor, while others have shown that Sprouty interferes with the MAPK cascade at the level of RAF activation. In addition to modulating the MAPK pathway in response to FGF stimulation, Sprouty itself appears to be subject to complex post-translational modification that regulates its activity and stability
The signal from unattached kinetochores is amplified through a Mad2 inhibitory signal that is propagated by the binding of Mad1 to the kinetochore, the association of Mad2 with Mad1, the conversion of Mad2 conformation to an inhibitory form through its association with Mad1 and finally the release of the inhibitory form of Mad2 from the kinetochore
Many hormones that affect individual physiological processes including the regulation of appetite, absorption, transport, and oxidation of foodstuffs influence energy metabolism pathways. While insulin mediates the storage of excess nutrients, glucagon is involved in the mobilization of energy resources in response to low blood glucose levels, principally by stimulating hepatic glucose output. Small doses of glucagon are sufficient to induce significant glucose elevations. These hormone-driven regulatory pathways enable the body to sense and respond to changed amounts of nutrients in the blood and demands for energy.Glucagon and Insulin act through various metabolites and enzymes that target specific steps in metabolic pathways for sugar and fatty acids. The processes responsible for the long-term control of fat synthesis and short term control of glycolysis by key metabolic products and enzymes are annotated in this module as six specific pathways:Pathway 1. Glucagon signalling in metabolic pathways: In response to low blood glucose, pancreatic alpha-cells release glucagon. The binding of glucagon to its receptor results in increased cAMP synthesis, and Protein Kinase A (PKA) activation.Pathway 2. PKA mediated phosphorylation:PKA phosphorylates key enzymes, e.g., 6-Phosphofructo-2-kinase /Fructose-2,6-bisphosphatase (PF2K-Pase) at serine 36, and regulatory proteins, e.g., Carbohydrate Response Element Binding Protein (ChREBP) at serine 196 and threonine 666.In brief, the binding of insulin to its receptor leads to increased protein phosphatase activity and to hydrolysis of cAMP by cAMP phosphodiesterase. These events counteract the regulatory effects of glucagon.Pathway 3: Insulin stimulates increased synthesis of Xylulose-5-phosphate (Xy-5-P). Activation of the insulin receptor results indirectly in increased Xy-5-P synthesis from Glyceraldehyde-3-phosphate and Fructose-6-phosphate. Xy-5-P, a metabolite of the pentose phosphate pathway, stimulates protein phosphatase PP2A.Pathway 4: AMP Kinase (AMPK) mediated response to high AMP:ATP ratio: In response to diet with high fat content or low energy levels, the cytosolic AMP:ATP ratio is increased. AMP triggers a complicated cascade of events. In this module we have annotated only the phosphorylation of ChREBP by AMPK at serine 568, which inactivates this transcription factor.Pathway 5: Dephosphorylation of key metabolic factors by PP2A: Xy-5-P activated PP2A efficiently dephosphorylates phosphorylated PF2K-Pase resulting in the higher output of F-2,6-P2 that enhances PFK activity in the glycolytic pathway. PP2A also dephosphorylates (and thus activates) cytosolic and nuclear ChREBP.Pathway 6: Transcriptional activation of metabolic genes by ChREBP: Dephosphorylated ChREBP activates the transcription of genes involved in glucose metabolism such as pyruvate kinase, and lipogenic genes such as acetyl-CoA carboxylase, fatty acid synthetase, acyl CoA synthase and glycerol phosphate acyl transferase. The illustration below summarizes this network of events. Black lines are metabolic reactions, red lines are negative regulatory events, and green lines are positive regulatory events (figure reused with permission from Veech (2003) - Copyright (2003) National Academy of Sciences, U.S.A.)
A member of the PP2A family of phosphatases dephosphorylates both cytosolic and nuclear forms of ChREBP (Carbohydrate Response Elemant Binding Protein). In the nucleus, dephosphorylated ChREBP complexes with MLX protein and binds to ChRE sequence elements in chromosomal DNA, activating transcription of genes involved in glycolysis and lipogenesis. The phosphatase is activated by Xylulose-5-phosphate, an intermediate of the pentose phosphate pathway (Kabashima et al. 2003). The rat enzyme has been purified to homogeneity and shown by partial amino acid sequence analysis to differ from previously described PP2A phosphatases (Nishimura and Uyeda 1995) - the human enzyme has not been characterized
Dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa (DARPP-32), was identified as a major target for dopamine and protein kinase A (PKA) in striatum. Recent advances now indicate that regulation DARPP-32 phosphorylation provides a mechanism for integrating information arriving at dopaminoceptive neurons, in multiple brain regions, via a variety of neurotransmitters, neuromodulators, neuropeptides, and steroid hormones. Activation of PKA or PKG stimulates DARPP-32 phosphorylation at Thr34, converting DARPP-32 into a potent inhibitor of protein phosphatase-1 (PP-1). DARPP-32 is also phosphorylated at Thr75 by Cdk5, converting DARPP-32 into an inhibitor of PKA. Thus, DARPP-32 has the unique property of being a dual-function protein, acting either as an inhibitor of PP-1 or of PKA
The beta-catenin destruction complex plays a key role in the canonical Wnt signaling pathway. In the absence of Wnt signaling, this complex controls the levels of cytoplamic beta-catenin. Beta-catenin associates with and is phosphorylated by the destruction complex. Phosphorylated beta-catenin is recognized and ubiquitinated by the SCF-beta TrCP ubiquitin ligase complex and is subsequently degraded by the proteasome (reviewed in Kimelman and Xu, 2006)
Degradation of beta-catenin is initiated following amino-terminal serine/threonine phosphorylation. Phosphorylation of B-catenin at S45 by CK1 alpha primes the subsequent sequential GSK-3-mediated phosphorylation at Thr41, Ser37 and Ser33 (Amit et al., 2002 ; Lui et al., 2002)
ERK/MAPK kinases have a number of targets within the nucleus, usually transcription factors or other kinases. The best known targets, ELK1, ETS1, ATF2, MITF, MAPKAPK2, MSK1, RSK1/2/3 and MEF2 are annotated here
MAP Kinases are inactivated by a family of protein named MAP Kinase Phosphatases (MKPs). They act through dephosphorylation of threonine and/or tyrosine residues within the signature sequence -pTXpY- located in the activation loop of MAP kinases (pT=phosphothreonine and pY=phosphotyrosine). MKPs are divided into three major categories depending on their preference for dephosphorylating; tyrosine, serine/threonine and both the tyrosine and threonine (dual specificity phoshatases or DUSPs). The tyrosine-specific MKPs include PTP-SL, STEP and HePTP, serine/threonine-specific MKPs are PP2A and PP2C, and many DUSPs acting on MAPKs are known. Activated MAP kinases trigger activation of transcription of MKP genes. Therefore, MKPs provide a negative feedback regulatory mechanism on MAPK signaling, by inactivating MAPKs via dephosphorylation, in the cytoplasm and the nucleus. Some MKPs are more specific for ERKs, others for JNK or p38MAPK
The activity of MASTL, also known as the Greatwall kinase (GWL), is necessary for the entry and progression of mitosis. MASTL is activated by phosphorylation of several key residues during mitotic entry. Phosphorylation on the serine residue S875 (S883 in Xenopus), likely through autophosphorylation (Blake-Hodek et al. 2012) appears to be critical (Vigneron et al. 2011). Several other sites, including putative CDK1 targets T194, T207 and T741, contribute to the full activation of MASTL (Yu et al. 2006, Blake-Hodek et al. 2012). Other kinases, such as PLK1 (Vigneron et al. 2011) and other MASTL phosphorylation sites may also be functionally important (Yu et al. 2006, Blake-Hodek et al. 2012).Activated MASTL phosphorylates ARPP19 and ENSA on serines S62 and S67, respectively, enabling them to bind to and inhibit the phosphatase activity of PP2A complexed with the regulatory subunit PPP2R2D (B55-delta). Inhibition of PP2A-PPP2R2D activity by ARPP19 or ENSA prevents dephosphorylation of CDK1 targets, hence allowing entry and maintenance of mitosis (Mochida et al. 2010, Gharbi-Ayachi et al. 2010, Burgess et al. 2010)
While sister chromatids resolve in prometaphase, separating along chromosomal arms, the cohesion of sister centromeres persists until anaphase. At the anaphase onset, the anaphase promoting complex/cyclosome (APC/C) ubiquitinates PTTG1 (securin), targeting it for degradation (Hagting et al. 2002). PTTG1 acts as an inhibitor of ESPL1 (known as separin i.e. separase). Hence, PTTG1 removal initiated by APC/C, enables ESPL1 to become catalytically active (Zou et al. 1999, Waizenegger et al. 2002). ESPL1 undergoes autoleavage (Waizenegger et al. 2002) and also cleaves RAD21 subunit of centromeric cohesin (Hauf et al. 2001). RAD21 cleavage promotes dissociation of cohesin complexes from sister centromeres, leading to separation of sister chromatids. Subsequent movement of sister chromatids to opposite poles of the mitotic spindle segregates replicated chromosomes to two daughter cells (Waizenegger et al. 2000, Hauf et al. 2001, Waizenegger et al. 2002)
The resolution of sister chromatids in mitotic prometaphase involves removal of cohesin complexes from chromosomal arms, with preservation of cohesion at centromeres (Losada et al. 1998, Hauf et al. 2001, Hauf et al. 2005). CDK1-mediated phosphorylation of cohesin-bound CDCA5 (Sororin) at threonine T159 provides a docking site for PLK1, enabling PLK1-mediated phosphorylation of cohesin subunits STAG2 (SA2) and RAD21 (Hauf et al. 2005, Dreier et al. 2011, Zhang et al. 2011). Further phosphorylation of CDCA5 by CDK1 results in dissociation of CDCA5 from cohesin complex, which restores the activity of WAPAL in removing STAG2-phosphorylated cohesin from chromosomal arms (Hauf et al. 2005, Gandhi et al. 2006, Kueng et al. 2006, Shintomi and Hirano 2006, Nishiyama et al. 2010, Zhang et al. 2011). At centromeres, kinetochore proteins shugoshins (SGOL1 and SGOL2) enable PP2A-B56 (also a kinetochore constituent) to dephosphorylate the STAG2 subunit of centromeric cohesin. Dephosphorylation of STAG2 enables maintenance of centromeric cohesion, thus preventing separation of sister chromatids until anaphase (Salic et al. 2004, Kitajima et al. 2004, Kitajima et al. 2005, Kitajima et al. 2006)
CTLA4 is one of the best studied inhibitory receptors of the CD28 superfamily. CTLA4 inhibits Tcell activation by reducing IL2 production and IL2 expression, and by arresting T cells at the G1 phase of the cell cycle. CTLA-4 expressed by a T cell subpopulation exerts a dominant control on the proliferation of other T cells, which limits autoreactivity. CTLA4 also blocks CD28 signals by competing for the ligands B71 and B72 in the limited space between T cells and antigenpresenting cells. Though the mechanism is obscure, CTLA4 may also propagate inhibitory signals that actively counter those produced by CD28. CTLA4 can also function in a ligand-independent manner.?CTLA-4 regulates the activation of pathogenic T cells by directly modulating T cell receptor signaling (i.e. TCR-zeta chain phosphorylation) as well as downstream biochemical signals (i.e. ERK activation). The cytoplasmic region of CTLA4 contains a tyrosine motif YVKM and a proline rich region. After TCR stimulation, it undergoes tyrosine phosphorylation by src kinases, inducing surface retention
Physiological concentrations (1g/L) of Low density lipoprotein (LDL) enhance platelet aggregation responses initiated by thrombin, collagen, and ADP. This enhancement involves the rapid phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) at Thr180 and Tyr182. The receptor for LDL is ApoER2, a splice variant of the classical ApoE receptor. ApoER2 stimulation leads to association of the Src family kinase Fgr which is probably responsible for subsequent phosphorylation of p38MAPK. This stimulation is transient because LDL also increases the activity of PECAM-1, which stimulates phosphatases that dephosphorylate p38MAPK
Upon stimulation with WNT ligand, AXIN and GSK3beta are recruited to the plasma membrane through interaction with DVL (Tamai et al, 2004; Mao et al, 2001; reviewed in He et al, 2004). Polymerization of membrane-associated DVL and GSK3beta- and CSNK1-mediated phosphorylation of LRP5/6 establish a feed-forward mechanism for enhanced membrane recruitment of AXIN upon WNT signaling (Tamai et al, 2004; Cong et al, 2004; Zeng et al, 2005; Bilic et al, 2007). In Xenopus oocytes, but not necessarily all sytems, AXIN is present in limiting concentrations and is considered rate limiting for the assembly of the destruction complex (Lee et al, 2003; Benchabane et al, 2008; Tan et al, 2012; reviewed in MacDonald et al, 2009). The recruitment of AXIN away from the destruction complex upon WNT stimulation effectively destabilizes the destruction complex and contributes to the accumulation of free beta-catenin (Kikuchi, 1999; Lee et al, 2003). AXIN association with the destruction complex is also regulated by phosphorylation. In the active destruction complex, AXIN is phosphorylated by GSK3beta; dephosphorylation by protein phosphatase 1 (PP1) or protein phosphatase 2A (PP2A) destabilizes the interaction of AXIN with the other components of the destruction complex and promotes its disassembly (Luo et al, 2007; Willert et al, 1999; Jho et al, 1999). Free AXIN is also subject to degradation by the 26S proteasome in a manner that depends on the poly-ADP-ribosylating enzymes tankyrase 1 and 2 (Huang et al, 2009)
GSK3beta is subject to in-frame missplicing in CML stem cells resulting in the production of mutant protein that lacks the AXIN and FRAT binding domains. Cells containing this mutant GSK3beta show elevated levels of nuclear beta-catenin and enhanced TCF-dependent reporter activity (Jamieson et al, 2008; Abrahamsson et al, 2009)
S33 mutations of beta-catenin interfere with GSK3 phosphorylation and result in stabilization and nuclear localization of the protein and enhanced WNT signaling (Groen et al, 2008; Nhieu et al, 1999; Clements et al, 2002; reviewed in Polakis, 2000). S33 mutations have been identified in cancers of the central nervous system, liver, endometrium and stomach, among others (reviewed in Polakis, 2000)
S37 mutations of beta-catenin interfere with GSK3 phosphorylation and stabilize the protein, resulting in enhanced WNT pathway signaling (Nhieu et al, 1999; Clements et al, 2002; reviewed in Polakis, 2000). S37 mutations have been identified in cancers of the brain, liver, ovary and large intestine, among others (reviewed in Polakis, 2000)
S45 mutants of beta-catenin have been identified in colorectal and hepatocellular carcinomas, soft tissue cancer and Wilms Tumors, among others (reviewed in Polakis, 2000). These mutations abolish the CK1alpha phosphorylation site of beta-catenin which acts as a critical priming site for GSK3 phosphorylation of T41( and subsequently S37 and S33) thus preventing its ubiquitin-mediated degradation (Morin et al, 1997; Amit et al, 2002)
T41 mutations of beta-catenin interfere with GSK3 phosphorylation and result in stabilization and nuclear accumulation of the protein (Moreno-Bueno et al, 2002; Taniguchi et al, 2002; reviewed in Polakis, 2012). T41 mutations have been identified in cancers of the liver and brain, as well as in the pituitary, endometrium, large intestine and skin, among others (reviewed in Polakis, 2000; Saito-Diaz et al, 2013)
Mutations in the APC tumor suppressor gene are common in colorectal and other cancers and cluster in the central mutation cluster region (MCR) of the gene (Miyoshi et al, 1992; Nagase and Nakamura, 1993; Dihlmann et al, 1999; reviewed in Bienz and Clevers, 2000). These mutations generally result in truncated proteins that destabilize the destruction complex and result in elevated WNT pathway activation (reviewed in Polakis, 2000)
Alterations in AXIN1 have been detected in a number of different cancers including liver and colorectal cancer and medullablastoma, among others (reviewed in Salahshor and Woodgett, 2005). Missense and nonsense mutations that disrupt or remove protein-protein interaction domains are common, and AXIN variants in cancers tend to disrupt the formation of a functional destruction complex (Satoh et al, 2000; Taniguchi et al, 2002; Webster et al, 2000; Shimizu et al, 2002)
AMER1/WTX is a known component of the destruction complex and interacts directly with beta-catenin through the C-terminal half (Major et al, 2007). siRNA depletion of AMER1 in mammalian cells stabilizes cellular beta-catenin levels and increases the expression of a beta-catenin-dependent reporter gene, suggesting that AMER1 is a tumor suppressor gene (Major et al, 2007; reviewed in Huff, 2011). Consistent with this, nonsense and missense mutations that truncate AMER1 and result in loss of the beta-catenin binding region have been identified in Wilms tumor, a pediatric kidney cancer (Ruteshouser et al, 2008; Wegert et al, 2009)
Formins are a family of proteins with 15 members in mammals, organized into 8 subfamilies. Formins are involved in the regulation of actin cytoskeleton. Many but not all formin family members are activated by RHO GTPases. Formins that serve as effectors of RHO GTPases belong to different formin subfamilies but they all share a structural similarity to Drosophila protein diaphanous and are hence named diaphanous-related formins (DRFs).
DRFs activated by RHO GTPases contain a GTPase binding domain (GBD) at their N-terminus, followed by formin homology domains 3, 1, and 2 (FH3, FH1, FH2) and a diaphanous autoregulatory domain (DAD) at the C-terminus. Most DRFs contain a dimerization domain (DD) and a coiled-coil region (CC) in between FH3 and FH1 domains (reviewed by Kuhn and Geyer 2014). RHO GTPase-activated DRFs are autoinhibited through the interaction between FH3 and DAD which is disrupted upon binding to an active RHO GTPase (Li and Higgs 2003, Lammers et al. 2005, Nezami et al. 2006). Since formins dimerize, it is not clear whether the FH3-DAD interaction is intra- or intermolecular. FH2 domain is responsible for binding to the F-actin and contributes to the formation of head-to-tail formin dimers (Xu et al. 2004). The proline-rich FH1 domain interacts with the actin-binding proteins profilins, thereby facilitating actin recruitment to formins and accelerating actin polymerization (Romero et al. 2004, Kovar et al. 2006).
Different formins are activated by different RHO GTPases in different cell contexts. FMNL1 (formin-like protein 1) is activated by binding to the RAC1:GTP and is involved in the formation of lamellipodia in macrophages (Yayoshi-Yamamoto et al. 2000) and is involved in the regulation of the Golgi complex structure (Colon-Franco et al. 2011). Activation of FMNL1 by CDC42:GTP contributes to the formation of the phagocytic cup (Seth et al. 2006). Activation of FMNL2 (formin-like protein 2) and FMNL3 (formin-like protein 3) by RHOC:GTP is involved in cancer cell motility and invasiveness (Kitzing et al. 2010, Vega et al. 2011). DIAPH1, activated by RHOA:GTP, promotes elongation of actin filaments and activation of SRF-mediated transcription which is inhibited by unpolymerized actin (Miralles et al. 2003). RHOF-mediated activation of DIAPH1 is implicated in formation of stress fibers (Fan et al. 2010). Activation of DIAPH1 and DIAPH3 by RHOB:GTP leads to actin coat formation around endosomes and regulates endosome motility and trafficking (Fernandez-Borja et al. 2005, Wallar et al. 2007). Endosome trafficking is also regulated by DIAPH2 transcription isoform 3 (DIAPH2-3) which, upon activation by RHOD:GTP, recruits SRC kinase to endosomes (Tominaga et al. 2000, Gasman et al. 2003). DIAPH2 transcription isoform 2 (DIAPH2-2) is involved in mitosis where, upon being activated by CDC42:GTP, it facilitates the capture of astral microtubules by kinetochores (Yasuda et al. 2004, Cheng et al. 2011). DIAPH2 is implicated in ovarian maintenance and premature ovarian failure (Bione et al. 1998). DAAM1, activated by RHOA:GTP, is involved in linking WNT signaling to cytoskeleton reorganization (Habas et al. 2001)
Mammals have three RAF isoforms, A, B and C, that are activated downstream of RAS and stimulate the MAPK pathway. Although CRAF (also known as RAF-1) was the first identified and remains perhaps the best studied, BRAF is most similar to the RAF expressed in other organisms. Notably, MAPK (ERK) activation is more compromised in BRAF-deficient cells than in CRAF or ARAF deficient cells (Bonner et al, 1985; Mikula et al, 2001, Huser et al, 2001, Mercer et al, 2002; reviewed in Leicht et al, 2007; Matallanas et al, 2011; Cseh et al, 2014). Consistent with its important role in MAPK pathway activation, mutations in the BRAF gene, but not in those for A- or CRAF, are associated with cancer development (Davies et al, 2002; reviewed in Leicht et al, 2007). ARAF and CRAF may have arisen through gene duplication events, and may play additional roles in MAPK-independent signaling (Hindley and Kolch, 2002; Murakami and Morrison, 2001).Despite divergences in function, all mammalian RAF proteins share three conserved regions (CRs) and each interacts with RAS and MEK proteins, although with different affinities. The N-terminal CR1 contains a RAS-binding domain (RBD) and a cysteine-rich domain (CRD) that mediate interactions with RAS and the phospholipid membrane. CR2 contains inhibitory phosphorylation sites that impact RAS binding and RAF activation, while the C-terminal CR3 contains the bi-lobed kinase domain with its activation loop, and an adjacent upstream \N-terminal acidic motif\ -S(S/G)YY in C- and A-RAF,respectively, and SSDD in B-RAF - that is required for RAF activation (Tran et al, 2005; Dhillon et al, 2002; Chong et al, 2001; Cutler et al, 1998; Chong et al, 2003; reviewed in Matallanas et al, 2011).Regulation of RAF activity involves multiple phosphorylation and dephosphorylation events, intramolecular conformational changes, homo- and heterodimerization between RAF monomers and changes to protein binding partners, including scaffolding proteins which bring pathway members together (reviewed in Matallanas et al, 2011; Cseh et al, 2014). The details of this regulation are not completely known and differ slightly from one RAF isoform to another. Briefly, in the inactive state, RAF phosphorylation on conserved serine residues in CR2 promote an interaction with 14-3-3 dimers, maintaining the kinase in a closed conformation. Upon RAS activation, these sites are dephosphorylated, allowing the RAF CRD and RBD to bind RAS and phospholipids, facilitating membrane recruitment. RAF activation requires homo- or heterodimerization, which promotes autophosphorylation in the activation loop of the receiving monomer. Of the three isoforms, only BRAF is able to initiate this allosteric activation of other RAF monomers (Hu et al, 2013; Heidorn et al, 2010; Garnett et al, 2005). This activity depends on negative charge in the N-terminal acidic region (NtA; S(S/G)YY or SSDD) adjacent to the kinase domain. In BRAF, this region carries permanent negative charge due to the presence of the two aspartate residues in place of the tyrosine residues of A- and CRAF. In addition, unique to BRAF, one of the serine residues of the NtA is constitutively phosphorylated. In A- and CRAF, residues in this region are subject to phosphorylation by activated MEK downstream of RAF activation, establishing a positive feedback loop and allowing activated A- and CRAF monomers to act as transactivators in turn (Hu et al, 2013; reviewed in Cseh et al, 2014). RAF signaling is terminated through dephosphorylation of the NtA region and phosphorylation of the residues that mediate the inhibitory interaction with 14-3-3, promoting a return to the inactive state (reviewed in Matallanas et al, 2011; Cseh et al, 2014)
The duration and extent of activated MAPK signaling is regulated at many levels through mechanisms that include phosphorylation and dephosphorylation, changes to protein interacting partners and subcellular localization (reviewed in Matallanas et al, 2011). Activated RAF proteins are subject to MAPK-dependent phosphorylation that promotes the subsequent dephosphorylation of the activation loop and NtA region, terminating RAF kinase activity. This dephosphorylation, catalyzed by PP2A and PP5, primes the RAF proteins for PKA or AKT-mediated phosphorylation of residues S259 and S621, restoring the 14-3-3 binding sites and returning the RAF proteins to the inactive state (von Kriegsheim et al, 2006; Dougherty et al, 2005; reviewed in Matallanas et al, 2011). The phosphorylated RAF1 NtA is also subject to additional regulation through binding to the PEBP1 protein, which promotes its dissociation from MAP2K substrates (Shin et al, 2009). Activated MAPK proteins also phosphorylate T292 of MAP2K1; this phosphorylation limits the activity of MAP2K1, and indirectly affects MAP2K2 activity through by modulating the activity of the MAP2K heterodimer (Catalanotti et al, 2009; reviewed in Matallanas et al, 2011).Dephosphorylation of MAPKs by the dual specificity MAPK phosphatases (DUSPs) plays a key role in limiting the extent of pathway activation (Owens et al, 2007; reviewed in Roskoski, 2012b). Class I DUSPs are localized in the nucleus and are induced by activation of the MAPK pathway, establishing a negative feedback loop, while class II DUSPs dephosphorylate cytoplasmic MAPKs (reviewed in Rososki, 2012b).MAPK signaling is also regulated by the RAS GAP-mediated stimulation of intrinsic RAS GTPase activity which returns RAS to the inactive, GDP bound state (reviewed in King et al, 2013)
In unstressed cells, TP53 (p53) has a short half-life as it undergoes rapid ubiquitination and proteasome-mediated degradation. The E3 ubiquitin ligase MDM2, which is a transcriptional target of TP53, plays the main role in TP53 protein down-regulation (Wu et al. 1993). MDM2 forms homodimers and homo-oligomers, but also functions as a heterodimer/hetero-oligomer with MDM4 (MDMX) (Sharp et al. 1999, Cheng et al. 2011, Huang et al. 2011, Pant et al. 2011). The heterodimers of MDM2 and MDM4 may be especially important for downregulation of TP53 during embryonic development (Pant et al. 2011).
The nuclear localization of MDM2 is positively regulated by AKT- or SGK1- mediated phosphorylation (Mayo and Donner 2001, Zhou et al. 2001, Amato et al. 2009, Lyo et al. 2010). Phosphorylation of MDM2 by CDK1 or CDK2 decreases affinity of MDM2 for TP53 (Zhang and Prives 2001). ATM and CHEK2 kinases, activated by double strand DNA breaks, phosphorylate TP53, reducing its affinity for MDM2 (Banin et al. 1998, Canman et al. 1998, Khanna et al. 1998, Chehab et al. 1999, Chehab et al. 2000). At the same time, ATM phosphorylates MDM2, preventing MDM2 dimerization (Cheng et al. 2009, Cheng et al. 2011). Both ATM and CHEK2 phosphorylate MDM4, triggering MDM2-mediated ubiquitination of MDM4 (Chen et al. 2005, Pereg et al. 2005). Cyclin G1 (CCNG1), transcriptionally induced by TP53, targets the PP2A phosphatase complex to MDM2, resulting in dephosphorylation of MDM2 at specific sites, which can have either a positive or a negative impact on MDM2 function (Okamoto et al. 2002).
In contrast to MDM2, E3 ubiquitin ligases RNF34 (CARP1) and RFFL (CARP2) can ubiquitinate phosphorylated TP53 (Yang et al. 2007).
In addition to ubiquitinating MDM4 (Pereg et al. 2005), MDM2 can also undergo auto-ubiquitination (Fang et al. 2000). MDM2 and MDM4 can be deubiquitinated by the ubiquitin protease USP2 (Stevenson et al. 2007, Allende-Vega et al. 2010). The ubiquitin protease USP7 can deubiquitinate TP53, but in the presence of DAXX deubiquitinates MDM2 (Li et al. 2002, Sheng et al. 2006, Tang et al. 2006).
The tumor suppressor p14-ARF, expressed from the CDKN2A gene in response to oncogenic or oxidative stress, forms a tripartite complex with MDM2 and TP53, sequesters MDM2 from TP53, and thus prevents TP53 degradation (Zhang et al. 1998, Parisi et al. 2002, Voncken et al. 2005).
For review of this topic, please refer to Kruse and Gu 2009
Phosphatidylinositol-5-phosphate (PI5P) may modulate PI3K/AKT signaling in several ways. PI5P is used as a substrate for production of phosphatidylinositol-4,5-bisphosphate, PI(4,5)P2 (Rameh et al. 1997, Clarke et al. 2008, Clarke et al. 2010, Clarke and Irvine 2013, Clarke et al. 2015), which serves as a substrate for activated PI3K, resulting in the production of PIP3 (Mandelker et al. 2009, Burke et al. 2011). The majority of PI(4,5)P2 in the cell, however, is produced from the phosphatidylinositol-4-phosphate (PI4P) substrate (Zhang et al. 1997, Di Paolo et al. 2002, Oude Weernink et al. 2004, Halstead et al. 2006, Oude Weernink et al. 2007). PIP3 is necessary for the activating phosphorylation of AKT. AKT1 can be deactivated by the protein phosphatase 2A (PP2A) complex that contains a regulatory subunit B56-beta (PPP2R5B) or B56-gamma (PPP2R5C). PI5P inhibits AKT1 dephosphorylation by PP2A through an unknown mechanism (Ramel et al. 2009). Increased PI5P levels correlate with inhibitory phosphorylation(s) of the PP2A complex. MAPK1 (ERK2) and MAPK3 (ERK1) are involved in inhibitory phosphorylation of PP2A, in a process that involves IER3 (IEX-1) (Letourneux et al. 2006, Rocher et al. 2007). It is uncertain, however, whether PI5P is in any way involved in ERK-mediated phosphorylation of PP2A or if it regulates another PP2A kinase
The dissolution of the nuclear membrane marks the beginning of the prometaphase. Kinetochores are created when proteins attach to the centromeres. Microtubules then attach at the kinetochores, and the chromosomes begin to move to the metaphase plate
Three D-type cyclins are essential for progression from G1 to S-phase. These D cyclins bind to and activate both CDK4 and CDK6. The formation of all possible complexes between the D-type cyclins and CDK4/6 is promoted by the proteins, p21(CIP1/WAF1) and p27(KIP1). The cyclin-dependent kinases are then activated due to phosphorylation by CAK. The cyclin dependent kinases phosphorylate the RB1 protein and RB1-related proteins p107 (RBL1) and p130 (RBL2). Phosphorylation of RB1 leads to release of activating E2F transcription factors (E2F1, E2F2 and E2F3). After repressor E2Fs (E2F4 and E2F5) dissociate from phosphorylated RBL1 and RBL2, activating E2Fs bind to E2F promoter sites, stimulating transcription of cell cycle genes, which then results in proper G1/S transition. The binding and sequestration of p27Kip may also contribute to the activation of CDK2 cyclin E/CDK2 cyclin A complexes at the G1/S transition (Yew et al., 2001)
Cell cycle progression is regulated by cyclin-dependent protein kinases at both the G1/S and the G2/M transitions. The G2/M transition is regulated through the phosphorylation of nuclear lamins and histones (reviewed in Sefton, 2001).The two B-type cyclins localize to different regions within the cell and are thought to have specific roles as CDK1-activating subunits (see Bellanger et al., 2007). Cyclin B1 is primarily cytoplasmic during interphase and translocates into the nucleus at the onset of mitosis (Jackman et al., 1995; Hagting et al., 1999). Cyclin B2 colocalizes with the Golgi apparatus and contributes to its fragmentation during mitosis (Jackman et al., 1995; Draviam et al., 2001)
The reactions of glycolysis (e.g., van Wijk and van Solinge 2005) convert glucose 6-phosphate to pyruvate. The entire process is cytosolic. Glucose 6-phosphate is reversibly isomerized to form fructose 6-phosphate. Phosphofructokinase 1 catalyzes the physiologically irreversible phosphorylation of fructose 6-phosphate to form fructose 1,6-bisphosphate. In six reversible reactions, fructose 1,6-bisphosphate is converted to two molecules of phosphoenolpyruvate and two molecules of NAD+ are reduced to NADH + H+. Each molecule of phosphoenolpyruvate reacts with ADP to form ATP and pyruvate in a physiologically irreversible reaction. Under aerobic conditions the NADH +H+ can be reoxidized to NAD+ via electron transport to yield additional ATP, while under anaerobic conditions or in cells lacking mitochondria NAD+ can be regenerated via the reduction of pyruvate to lactate