Characterization of equine cytochrome P450: role of CYP3A in the metabolism of diazepam.

Research on drug metabolism and pharmacokinetics in large animal species including the horse is scarce because of the challenges in conducting in vivo studies. The metabolic reactions catalyzed by cytochrome P450s (CYPs) are central to drug pharmacokinetics. This study elucidated the characteristics of equine CYPs using diazepam (DZP) as a model compound as this drug is widely used as an anesthetic and sedative in horses, and is principally metabolized by CYPs. Diazepam metabolic activities were measured in vitro using horse and rat liver microsomes to clarify the species differences in enzyme kinetic parameters of each metabolite (temazepam [TMZ], nordiazepam [NDZ], p-hydroxydiazepam [p-OH-DZP], and oxazepam [OXZ]). In both species microsomes, TMZ was the major metabolite, but the formation rate of p-OH-DZP was significantly less in the horse. Inhibition assays with a CYP-specific inhibitors and antibody suggested that CYP3A was the main enzyme responsible for DZP metabolism in horse. Four recombinant equine CYP3A isoforms expressed in Cos-7 cells showed that CYP3A96, CYP3A94, and CYP3A89 were important for TMZ formation, whereas CYP3A97 exhibited more limited activity. Phylogenetic analysis suggested diversification of CYP3As in each mammalian order. Further study is needed to elucidate functional characteristics of each equine CYP3A isoform for effective use of diazepam in horses.

Expression of Cryptosporidium parvum thioredoxin peroxidase in COS-7 cells confers radioprotection.

Cryptosporidium parvum is one of the most radioresistant organisms identified to date. In a previous study, we found that thioredoxin peroxidase (CpTPx) was significantly upregulated in this species following exposure to high dose (10 kGy) of γ-irradiation. To assess the potential of CpTPx to confer radioprotection in mammalian cells, it was expressed in COS-7 African green monkey kidney cells (CpTPx-COS7). For comparison, the thioredoxin peroxidase of Cryptosporidium muris (CmTPx) was also expressed in these cells (CmTPx-COS7 cells), which has been confirmed to have lesser antioxidant activity than CpTPx in the previous study. Notably, the survival rates of CpTPx-COS7 cells were significantly higher (12-22%) at 72 h after 8 Gy irradiation than CmTPx-COS7 or non-transfected COS-7 (ntCOS-7) counterparts. In addition, CpTPx revealed a 50% of ROS reduction in irradiated CpTPx-COS7 cells, while γ-H2AX DNA damage marker expression was not significantly changed. Furthermore, the amount of apoptosis only increased to about 120% after 2-8 Gy irradiation compared to 200-300% increase observed in ntCOS-7 cells. CmTPx was shown to have antioxidant and DNA damage protection activities; however, these activities were always lower than those of CpTPx. These results suggest that the potent antioxidant and protective activities of CpTPx are well conserved in this cell-based system and that CpTPx contributed to the radioprotection of mammalian cells through its exceptional antioxidant activity.

Promising results of the chaperone effect caused by imino sugars and aminocyclitol derivatives on mutant glucocerebrosidases causing Gaucher disease.

Gaucher disease is an autosomal recessive disorder. It is characterized by the accumulation of glucosylceramide in lysosomes of mononuclear phagocyte system, attributable to acid beta-glucosidase deficiency. The main consequences of this disease are hepatosplenomegaly, skeletal lesions and, sometimes, neurological manifestations. At sub-inhibitory concentrations, several competitive inhibitors act as chemical chaperones by inducing protein stabilization and increasing enzymatic activity. Here we tested two iminosugars (NB-DNJ and NN-DNJ) and four aminocyclitols with distinct degrees of lipophilicity as pharmacological chaperones for glucocerebrosidase (GBA). We report an increase in the activity of GBA using NN-DNJ, NB-DNJ and aminocyclitol 1 in stably transfected cell lines, and an increment with NN-DNJ and aminocyclitol 4 in patient fibroblasts. These results on specific mutations validate the use of chemical chaperones as a therapeutic approach for Gaucher disease. However, the development and analysis of new compounds is required in order to find more effective therapeutic agents that are active on a broader range of mutations.

The mixed-lineage kinase DLK undergoes Src-dependent tyrosine phosphorylation and activation in cells exposed to vanadate or platelet-derived growth factor (PDGF).

Some data in the literature suggest that serine/threonine phosphorylation is required for activation of the mixed-lineage kinases (MLKs), a subgroup of mitogen-activated protein kinase kinase kinases (MAPKKKs). In this report, we demonstrate that the MLK family member DLK is activated and concurrently tyrosine-phosphorylated in cells exposed to the protein tyrosine phosphatase inhibitor vanadate. Tyrosine phosphorylation appears crucial for activation as incubation of vanadate-activated DLK molecules with a tyrosine phosphatase substantially reduced DLK enzymatic activity. Interestingly, the effects of vanadate on DLK are completely blocked by treatment with a Src family kinase inhibitor, PP2, or the expression of short hairpin RNA (shRNA) directed against Src. DLK also fails to undergo vanadate-stimulated tyrosine phosphorylation and activation in fibroblasts which lack expression of Src, Yes and Fyn, but reintroduction of wild-type Src or Fyn followed by vanadate treatment restores this response. In addition to vanadate, stimulation of cells with platelet-derived growth factor (PDGF) also induces tyrosine phosphorylation and activation of DLK by a Src-dependent mechanism. DLK seems important for PDGF signaling because its depletion by RNA interference substantially reduces PDGF-stimulated ERK and Akt kinase activation. Thus, our findings suggest that Src-dependent tyrosine phosphorylation of DLK may be important for regulation of its activity, and they support a role for DLK in PDGF signaling.

Inositol 5'-phosphatase, SHIP1 interacts with phospholipase C-gamma1 and modulates EGF-induced PLC activity.

Phospholipase C-gamma1, containing two SH2 and one SH3 domains which participate in the interaction between signaling molecules, plays a significant role in the growth factor-induced signal transduction. However, the role of the SH domains in the growth factor-induced PLC-gamma1 regulation is unclear. By peptide-mass fingerprinting analysis, we have identified SHIP1 as the binding protein for the SH3 domain of PLC-gamma1. SHIP1 was co-immunoprecipitated with PLC-gamma1 and potentiated EGF-induced PLC-gamma1 activation. However, inositol 5'-phosphatase activity of SHIP1 was not required for the potentiation of EGF-induced PLC-gamma1 activation. Taken together, these results suggest that SHIP1 may function as an adaptor protein which can potentiate EGF-induced PLC-gamma1 activation without regards to its inositol 5'-phosphatase activity.

Lysophospholipid generation and phosphatidylglycerol depletion in phospholipase A(2)-mediated surfactant dysfunction.

Pulmonary surfactant's complex mixture of phospholipids and proteins reduces the work of breathing by lowering alveolar surface tension during respiration. One mechanism of surfactant damage appears to be the hydrolysis of phospholipid by phospholipases activated in the inflamed lung. Humans have several candidate secretory phospholipase A(2) (sPLA(2)) enzymes in lung cells and infiltrating leukocytes that could damage extracellular surfactant. We considered two mechanisms of surfactant disruption by five human sPLA(2)s, including generation of lysophospholipids and the depletion of specific phospholipids. All five sPLA(2)s studied ultimately caused surfactant dysfunction. Each enzyme exhibited a different pattern of hydrolysis of surfactant phospholipids. Phosphatidylcholine, the major phospholipid in surfactant and the greatest potential source for generation of lysophospholipids, was susceptible to hydrolysis by group IB, group V, and group X sPLA(2)s, but not group IIA or IID. Group IIA hydrolyzed both phosphatidylethanolamine and phosphatidylglycerol, whereas group IID was active against only phosphatidylglycerol. Thus, with groups IB and X, the generation of lysophospholipids corresponded with surfactant dysfunction. However, hydrolysis of and depletion of phosphatidylglycerol had a greater correlation with surfactant dysfunction for groups IIA and IID. Surfactant dysfunction caused by group V sPLA(2) is less clear and may be the combined result of both mechanisms.

Functional studies of human intestinal alkaline sphingomyelinase by deglycosylation and mutagenesis.

Intestinal alk-SMase (alkaline sphingomyelinase) is an ectoenzyme related to the NPP (nucleotide phosphodiesterase) family. It has five potential N-glycosylation sites and predicated transmembrane domains at both the N- and C-termini. The amino acid residues forming the two metal-binding sites in NPP are conserved, and those of the active core are modified. We examined the functional changes of the enzyme induced by deglycosylation and mutagenesis. Treating alk-SMase cDNA-transfected COS-7 cells with tunicamycin rendered the expressed enzyme completely inactive. Mutations of the five potential N-glycosylation sites individually and in combination showed that these sites were all glycosylated and deficient glycosylation decreased the enzyme activity. Immunogold labelling showed that the wild-type enzyme was mainly located in the plasma membrane, whereas the C-terminal domain-truncated enzyme was released into the medium. Deglycosylation blocked the release of the enzyme that accumulated in endosome-like structures. The enzyme activity was also decreased by mutations of the residues forming the putative metal-binding sites and the active core. Substitution of the active core sequence with that of NPP or mutation of T75 in the core abolished the enzyme activity against sphingomyelin but failed to render the enzyme NPP active. Our results indicate that alk-SMase activity is severely affected by defective N-glycosylation and structural alterations of the putative metal-binding sites and the predicted active core.

Regulation of adenylate cyclase type VIII splice variants by acute and chronic Gi/o-coupled receptor activation.

We previously reported that acute agonist activation of G(i/o)-coupled receptors inhibits adenylate cyclase (AC) type VIII activity, whereas agonist withdrawal following chronic activation of these receptors induces AC-VIII superactivation. Three splice variants of AC-VIII have been identified, which are called AC-VIII-A, -B and -C (with AC-VIII-B missing the glycosylation domain and AC-VIII-C lacking most of the C1b area). We report here that AC-VIII-A and -B, but not -C, are inhibited by acute mu-opioid and dopaminergic type D2 receptor activation, indicating that the C1b area of AC-VIII has an important role in AC inhibition by G(i/o)-coupled receptor activation. On the other hand the glycosylation sites in AC-VIII did not play a role in AC-VIII regulation. Although AC-VIII-A and -C differed in their capacity to be inhibited by acute agonist exposure, agonist withdrawal after prolonged treatment led to a similar superactivation of all three splice variants, with no significant change in AC-VIII expression. AC-VIII superactivation was not affected by pre-incubation with a cell permeable cAMP analogue, indicating that the superactivation does not depend on the agonist-induced reduction in cAMP levels. The superactivated AC-VIII-A, -B and -C were similarly re-inhibited by re-application of agonist (morphine or quinpirole), returning the activity to control levels. These results demonstrate marked differences in the agonist inhibition of the AC-VIII splice variants before, but not after, superactivation.

Inositol 5'-phosphatase, SHIP1 interacts with phospholipase C-gamma1 and modulates EGF-induced PLC activity

Phospholipase C-gamma1, containing two SH2 and one SH3 domains which participate in the interaction between signaling molecules, plays a significant role in the growth factor-induced signal transduction. However, the role of the SH domains in the growth factor-induced PLC-gamma1 regulation is unclear. By peptide-mass fingerprinting analysis, we have identified SHIP1 as the binding protein for the SH3 domain of PLC-gamma1. SHIP1 was co-immunoprecipitated with PLC-gamma1 and potentiated EGF-induced PLC-gamma1 activation. However, inositol 5'-phosphatase activity of SHIP1 was not required for the potentiation of EGF-induced PLC-gamma1 activation. Taken together, these results suggest that SHIP1 may function as an adaptor protein which can potentiate EGF-induced PLC-gamma1 activation without regards to its inositol 5'-phosphatase activity.

Transactivation of peroxisome proliferator-activated receptor alpha by green tea extracts.

Tea is a popular beverage. Recently, green tea was reported to increase the number of peroxisomes in rats. In this study, to find out whether the green tea-induced proliferation of peroxisomes is mediated by PPARalpha , a transient transfection assay was carried out to investigate the interactions of tea extracts (green tea, black tea,oolong tea and doongule tea) and tea components (epigallocatechin gallate, epigallocatechin, epicatechin gallate, epicatechin and gallic acid), with mouse cloned PPARalpha . Green tea and black tea extracts, and epigallocatechin gallate, a major component of fresh green tea leaves, increased the activation of PPAalpha 1.5-2 times compared with the control. It is suggested that the green tea induced-peroxisomal proliferation may be mediated through the transactivation of PPARalpha and that epigallocatechin gallate may be an effective component of green tea leaves. This would account for the increase in the number of peroxisomes and the activity of peroxisomal enzymes previously reported. However, black tea, a fully fermented product, had a stronger effect than oolong tea extract. These results also suggest, that in addition to epigallocatechin gallate, green tea leaves may possess some active chemicals newly produced as a result of the fermentation process, which act on PPARalpha like other peroxisome proliferators.

A novel aspartylglucosaminuria mutation affects translocation of aspartylglucosaminidase.

The AGA gene is mutated in patients with aspartylglucosaminuria (AGU), a lysosomal storage disease enriched in the Finnish population. The disease mechanism of AGU and the biochemistry and cell biology of the lysosomal aspartylglucosaminidase (AGA) enzyme are well characterized. Here, we have investigated a novel AGU mutation found in a Finnish patient. The mutation was detected as a compound heterozygote with the Finnish major mutation in the other allele. The novel point mutation, c.44T>G, causes the L15R amino acid substitution in the signal sequence of the AGA enzyme. The mutated AGA enzyme was here analyzed by over expression in BHK and COS-1 cells. The L15R AGA protein was only faintly detectable by immunofluorescence analysis and observed in the endoplasmic reticulum. Metabolic labeling and immunoprecipitation revealed only a small amount of AGA polypeptides but the specific activity of the mutant enzyme was surprisingly high, 37% of the wild type. The amino acid substitution probably affects translocation of AGA polypeptides by altering a critical hydrophobic core structure of the signal sequence. It appears that the small amounts of active enzyme are not able to reach the lysosomes thus explaining the development of AGU disease in the patient.

Characterization of a novel low-molecular-mass dual-specificity phosphatase-3 (LDP-3) that enhances activation of JNK and p38.

We have isolated a mouse cDNA for a novel dual-specificity phosphatase designated LDP-3 (low-molecular-mass dual-specificity phosphatase 3). The 450 bp open reading frame encodes a protein of 150 amino acids with a predicted molecular mass of 16 kDa. Northern blot and reverse transcription-PCR analyses show that LDP-3 transcripts are expressed in almost all mouse tissues examined. In vitro analyses using several substrates and inhibitors indicate that LDP-3 possesses intrinsic dual-specificity phosphatase activity. When expressed in mammalian cells, LDP-3 protein is localized mainly to the apical submembrane area. Forced expression of LDP-3 does not alter activation of ERK (extracellular-signal-regulated kinase), but rather enhances activation of JNK (c-Jun N-terminal kinase) and p38 and their respective upstream kinases MKK4 (mitogen-activated protein kinase kinase 4) and MKK6 in cells treated with 0.4 M sorbitol. By screening with a variety of stimuli, we found that LDP-3 specifically enhances the osmotic stress-induced activation of JNK and p38.

Delta-catenin/NPRAP (neural plakophilin-related armadillo repeat protein) interacts with and activates sphingosine kinase 1.

Sphingosine kinase (SPHK) is a key enzyme catalysing the formation of sphingosine 1-phosphate (SPP), a lipid messenger that is implicated in the regulation of a wide variety of important cellular events acting through intracellular, as well as extracellular, mechanisms. However, the molecular mechanism of intracellular actions of SPP remains unclear. Here, we have identified delta-catenin/NPRAP (neural plakophilin-related armadillo repeat protein) as a potential binding partner for SPHK1 by yeast two-hybrid screening. From co-immunoprecipitation analyses, the C-terminal portion of delta-catenin/NPRAP containing the seventh to tenth armadillo repeats was found to be required for interaction with SPHK1. Endogenous delta-catenin/NPRAP was co-localized with endogenous SPHK1 and transfected delta-catenin/NPRAP was co-localized with transfected SPHK1 in dissociated rat hippocampal neurons. MDCK (Madin-Darby canine kidney) cells stably expressing delta-catenin/NPRAP contained elevated levels of intracellular SPP. In a purified system delta-catenin/NPRAP stimulated SPHK1 in a dose-dependent manner. Furthermore, delta-catenin/NPRAP-induced increased cell motility in MDCK cells was completely inhibited by dimethylsphingosine, a specific inhibitor of SPHK1. These results strongly suggest that at least some of delta-catenin/NPRAP functions, including increased cell motility, are mediated by an SPHK-SPP signalling pathway.

p90 ribosomal S6 kinase 2 is associated with and dephosphorylated by protein phosphatase 2Cdelta.

RSK2 (p90 ribosomal S6 kinase 2) is activated via the ERK (extracellular-signal-regulated kinase) pathway by phosphorylation on four sites: Ser227 in the activation loop of the N-terminal kinase domain, Ser369 in the linker, Ser386 in the hydrophobic motif and Thr577 in the C-terminal kinase domain of RSK2. In the present study, we demonstrate that RSK2 is associated in vivo with PP2Cdelta (protein phosphatase 2Cdelta). In epidermal growth factorstimulated cells, RSK2 is partially dephosphorylated on all four sites in an Mn2+-dependent manner, leading to reduced protein kinase activity. Furthermore, PP2Cd is phosphorylated by ERK on Thr315 and Thr333 in the catalytic domain. Mutation of Thr315 and Thr333 to alanine in a catalytically inactive mutant PP2Cdelta (H154D) (His154-->Asp) increases the association with RSK2 significantly, whereas mutation to glutamate, mimicking phosphorylation, reduces the binding of RSK2. The domains of interaction are mapped to the N-terminal extension comprising residues 1-71 of PP2Cd and the N-terminal kinase domain of RSK2. The interaction is specific, since PP2Cd associates with RSK1-RSK4, MSK1 (mitogen- and stress-activated kinase 1) and MSK2, but not with p70 S6 kinase or phosphoinositide-dependent kinase 1. We conclude that RSK2 is associated with PP2Cd in vivo and is partially dephosphorylated by it, leading to reduced kinase activity.

ERK5 is targeted to myocyte enhancer factor 2A (MEF2A) through a MAPK docking motif.

One critical component in determining the specificity, and efficiency of MAPK (mitogen-activated protein kinase) substrate phophorylation is the presence of distinct docking domains in the substrate proteins. Docking domains have been shown to be important for the activities of members of the ERK (extracellular-signal-regulated kinase), JNK (c-Jun N-terminal kinase) and p38 subfamilies of MAPKs towards their substrates. Here, we demonstrate that docking domains also play an important role in ERK5-mediated substrate phosphorylation. The presence of a docking domain promotes both phosphorylation of myocyte enhancer factor, MEF2A, in vitro and its activation in vivo by ERK5. Mutational analysis of the MEF2A docking domain demonstrates that the specificity determinants for ERK5 are similar to those observed with members of the p38 subfamily. A docking domain recognized by ERK5 can direct ERK5 to activate heterologous substrates. Deletion analysis demonstrates that as with other MAPKs, it is the catalytic domain of ERK5 that recognizes the docking domain. Our data therefore extend previous observations on other MAPKs and demonstrate that the requirement for specific docking domains in promoting MAPK action towards substrates is a general property of MAPKs.

Regulation of Cyp2a5 transcription in mouse primary hepatocytes: roles of hepatocyte nuclear factor 4 and nuclear factor I.

The cytochrome P4502a5 (Cyp2a5) gene is expressed principally in liver and olfactory mucosa. In the present study, the transcriptional mechanisms of hepatocyte-specific expression of Cyp2a5 were studied in mouse primary hepatocytes. The Cyp2a5 5'-flanking region -3033 to +10 was cloned in front of a luciferase reporter gene and transfected into hepatocytes. Deletion analysis revealed two major activating promoter regions localized at proximal 271 bp and at a more distal area from -3033 to -2014 bp. The proximal activation region was characterized further by DNase I footprinting, and a single clear footprint was detected in the studied area centred over a sequence similar to the NF-I (nuclear factor I)-binding site. The binding of NF-I was confirmed using an EMSA (electrophoretic mobility-shift assay). A putative HNF-4 (hepatocyte nuclear factor 4)-binding site was localized at the proximal promoter by computer analysis of the sequence, and HNF-4alpha was shown to interact with the site using an EMSA. The functional significance of HNF-4 and NF-I binding to the Cyp2a5 promoter was evaluated by site-directed mutagenesis of the binding motifs in reporter constructs. Both mutations strongly decreased transcriptional activation by the Cyp2a5 promoter in primary hepatocytes, and double mutation almost completely abolished transcriptional activity. Also, the functionality of the distal activation region was found to be dependent on the intact HNF-4 and NF-I sites at the proximal promoter. In conclusion, these results indicate that HNF-4 and NF-I play major roles in the constitutive regulation of hepatic expression of Cyp2a5.

Regulation of the MST1 kinase by autophosphorylation, by the growth inhibitory proteins, RASSF1 and NORE1, and by Ras.

MST1 (mammalian Sterile20-like 1) and MST2 are closely related Class II GC (protein Ser/Thr) kinases that initiate apoptosis when transiently overexpressed in mammalian cells. In the present study, we show that recombinant MST1/2 undergo a robust autoactivation in vitro, mediated by an intramolecular autophosphorylation of a single site [MST1(Thr183)/MST2(Thr180)] on the activation loop of an MST dimer. Endogenous full-length MST1 is activated by a variety of stressful stimuli, accompanied by the secondary appearance of a 36 kDa Thr183-phosphorylated, caspase-cleaved catalytic fragment. Recombinant MST1 exhibits only 2-5% activation during transient expression; endogenous MST1 in the cycling HeLa or KB cells has a similar low fractional activation, but 2 h incubation with okadaic acid (1 mM) results in 100% activation. Endogenous MST1 immunoprecipitated from KB cells is specifically associated with substoichiometric amounts of the growth inhibitory polypeptides RASSF1A and NORE1A (novel Ras effector 1A; a Ras-GTP-binding protein). Co-expression of RASSF1A, RASSF1C, NORE1A and NORE1B with MST1 markedly suppresses MST1(Thr183) phosphorylation in vivo and abolishes the ability of MST1 to undergo Mg-ATP-mediated autoactivation in vitro; direct addition of purified NORE1A in vitro also inhibits MST1 activation. In contrast, co-transfection of MST1 with NORE1A modified by the addition of a C-terminal CAAX motif results in a substantial increase in MST1(Thr183) phosphorylation, as does fusion of a myristoylation motif directly on to the MST1 N-terminus. Moreover, MST1 polypeptides, bound via wild-type NORE1A to Ras(G12V) (where G12V stands for Gly12Val), exhibit higher Thr183 phosphorylation compared with MST1 bound to NORE1A alone. Nevertheless, serum stimulation of KB cells does not detectably increase the activation state of endogenous MST1 or MST2 despite promoting the recruitment of the endogenous NORE1-MST1 complex to endogenous Ras. We propose that the NORE1/RASSF1 polypeptides, in addition to their role in maintaining the low activity of MST1 in vivo, direct MST1 to sites of activation and perhaps co-localization with endogenous substrates.

Murine cystathionine gamma-lyase: complete cDNA and genomic sequences, promoter activity, tissue distribution and developmental expression.

Cystathionine gamma-lyase (CSE) is the last key enzyme in the trans-sulphuration pathway for biosynthesis of cysteine from methionine. Cysteine could be provided through diet; however, CSE has been shown to be important for the adequate supply of cysteine to synthesize glutathione, a major intracellular antioxidant. With a view to determining physiological roles of CSE in mice, we report the sequence of a complete mouse CSE cDNA along with its associated genomic structure, generation of specific polyclonal antibodies, and the tissue distribution and developmental expression patterns of CSE in mice. A 1.8 kb full-length cDNA containing an open reading frame of 1197 bp, which encodes a 43.6 kDa protein, was isolated from adult mouse kidney. A 35 kb mouse genomic fragment was obtained by lambda genomic library screening. It contained promoter regions, 12 exons, ranging in size from 53 to 579 bp, spanning over 30 kb, and exon/intron boundaries that were conserved with rat and human CSE. The GC-rich core promoter contained canonical TATA and CAAT motifs, and several transcription factor-binding consensus sequences. The CSE transcript, protein and enzymic activity were detected in liver, kidney, and, at much lower levels, in small intestine and stomach of both rats and mice. In developing mouse liver and kidney, the expression levels of CSE protein and activity gradually increased with age until reaching their peak value at 3 weeks of age, following which the expression levels in liver remained constant, whereas those in kidney decreased significantly. Immunohistochemical analyses revealed predominant CSE expression in hepatocytes and kidney cortical tubuli. These results suggest important physiological roles for CSE in mice.

The T-cell protein tyrosine phosphatase is phosphorylated on Ser-304 by cyclin-dependent protein kinases in mitosis.

Two alternatively spliced forms of the human protein tyrosine phosphatase TCPTP (T-cell protein tyrosine phosphatase) exist: a 48 kDa form that is targeted to the endoplasmic reticulum (TC48) and a shorter 45 kDa form that is targeted to the nucleus (TC45). In this study we have identified Ser-304 (Phe301-Asp-His-Ser304-Pro-Asn-Lys307) as a major TCPTP phosphory-lation site and demonstrate that TC45, but not TC48, is phosphorylated on this site in vivo. Phosphorylation of TC45 on Ser-304 was cell cycle-dependent, and increased as cells progressed from G2 into mitosis, but subsided upon mitotic exit. Ser-304 phosphorylation was increased when cells were arrested in mitosis by microtubule poisons such as nocodazole, but remained unaltered when cells were arrested at the G2/M checkpoint by adriamycin. Phosphorylation of Ser-304 did not alter significantly the phosphatase activity or the protein stability of TC45, and had no apparent effect on TC45 localization. Ser-304 phosphorylation was ablated when cells were treated with the CDK (cyclin-dependent protein kinase) inhibitors roscovitine or SU9516, but remained unaltered when ERK1/2 activation was inhibited with the MEK (mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase) inhibitor PD98059. In addition, recombinant CDKs, but not the Polo-like kinase Plk1, phosphorylated Ser-304 in vitro. Our studies identify Ser-304 as a major phosphorylation site in human TCPTP, and the TC45 variant as a novel mitotic CDK substrate.

Noonan syndrome-associated SHP2/PTPN11 mutants cause EGF-dependent prolonged GAB1 binding and sustained ERK2/MAPK1 activation.

Noonan syndrome is a developmental disorder with dysmorphic facies, short stature, cardiac defects, and skeletal anomalies, which can be caused by missense PTPN11 mutations. PTPN11 encodes Src homology 2 domain-containing tyrosine phosphatase 2 (SHP2 or SHP-2), a protein tyrosine phosphatase that acts in signal transduction downstream to growth factor, hormone, and cytokine receptors. We compared the functional effects of three Noonan syndrome-causative PTPN11 mutations on SHP2's phosphatase activity, interaction with a binding partner, and signal transduction. All SHP2 mutants had significantly increased basal phosphatase activity compared to wild type, but that activity varied significantly between mutants and was further increased after epidermal growth factor stimulation. Cells expressing SHP2 mutants had prolonged extracellular signal-regulated kinase 2 activation, which was ligand-dependent. Binding of SHP2 mutants to Grb2-associated binder-1 was increased and sustained, and tyrosine phosphorylation of both proteins was prolonged. Coexpression of Grb2-associated binder-1-FF, which lacks SHP2 binding motifs, blocked the epidermal growth factor-mediated increase in SHP2's phosphatase activity and resulted in a dramatic reduction of extracellular signal-regulated kinase 2 activation. Taken together, these results document that Noonan syndrome-associated PTPN11 mutations increase SHP2's basal phosphatase activity, with greater activation when residues directly involved in binding at the interface between the N-terminal Src homology 2 and protein tyrosine phosphatase domains are altered. The SHP2 mutants prolonged signal flux through the RAS/mitogen-activated protein kinase (ERK2/MAPK1) pathway in a ligand-dependent manner that required docking through Grb2-associated binder-1 (GAB1), leading to increased cell proliferation.