Identification of p130(cas) as a substrate for the cytosolic protein tyrosine phosphatase PTP-PEST.

PTP-PEST is a ubiquitously expressed, cytosolic, mammalian protein tyrosine phosphatase (PTP) which exhibits high specific activity in vitro. We have investigated the substrate specificity of PTP-PEST by a novel substrate-trapping approach in combination with in vitro dephosphorylation experiments. We initially identified a prominent 130-kDa tyrosine-phosphorylated protein in pervanadate-treated HeLa cell lysates which was preferentially dephosphorylated by PTP-PEST in vitro. In order to identify this potential substrate, mutant (substrate-trapping) forms of PTP-PEST were generated which lack catalytic activity but retain the ability to bind substrates. These mutant proteins associated in stable complexes exclusively with the same 130-kDa protein, which was identified as p130(cas) by immunoblotting. This exclusive association was observed in lysates from several cell lines and in transfected COS cells, but was not observed with other members of the PTP family, strongly suggesting that p130(cas) represents a major physiologically relevant substrate for PTP-PEST. Our studies suggest potential roles for PTP-PEST in regulation of p130(cas) function. These functions include mitogen- and cell adhesion-induced signalling events and probable roles in transformation by various oncogenes. These results provide the first demonstration of a PTP having an inherently restricted substrate specificity in vitro and in vivo. The methods used to identify p130(cas) as a specific substrate for PTP-PEST are potentially applicable to any PTP and should therefore prove useful in determining the physiological substrates of other members of the PTP family.     

Contingent phosphorylation/dephosphorylation provides a mechanism of molecular memory in WASP

Cells can retain information about previous stimuli to produce distinct future responses. The biochemical mechanisms by which this is achieved are not well understood. The Wiskott-Aldrich syndrome protein (WASP) is an effector of the Rho-family GTPase Cdc42, whose activation leads to stimulation of the actin nucleating assembly, Arp2/3 complex. We demonstrate that efficient phosphorylation and dephosphorylation of WASP at Y291 are both contingent on binding to activated Cdc42. Y291 phosphorylation increases the basal activity of WASP toward Arp2/3 complex and enables WASP activation by new stimuli, SH2 domains of Src-family kinases. The requirement for contingency in both phosphorylation and dephosphorylation enables long-term storage of information by WASP following decay of GTPase signals. This biochemical circuitry allows WASP to respond to the levels and timing of GTPase and kinase signals. It provides mechanisms to specifically achieve transient or persistent actin remodeling, as well as long-lasting potentiation of actin-based responses to kinases.     

A synthetic peptide derived from the non-structural protein 3 of hepatitis C virus serves as a specific substrate for PKC

A synthetic peptide corresponding to the amino acid sequence Arg1487-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Arg-Gly-Ile-Tyr-Arg1500 of the hepatitis C virus (HCV) polyprotein was found to be a selective substrate for protein kinase C (PKC). In the presence of Ca2+, TPA and phospholipid, PKC phosphorylates the peptide [termed HCV(1487-1500)] with a Km of 11 microM and Vmax of 24 micromol x min(-1) x mg(-1). HCV(1487-1500) acts as a competitive inhibitor of PKC towards other peptide or protein substrates and inhibits the kinase activity with an IC50 corresponding to the Km values measured for the substrates. N- or C-terminally deleted analogs of HCV(1487-1500) did not show inhibitory effects and were only marginally or not phosphorylatable. We designed an additional peptide in which the tyrosine residue was replaced by phenylalanine ([Phe1499]HCV(1487-1500)). This peptide was neither phosphorylated by other serine/threonine kinases tested nor by whole cell extracts prepared from PKC-depleted cells. [Phe1499]HCV(1487-1500) was used to monitor the TPA-induced translocation of PKC activity to the particulate fraction in JB6 cells. The use of SDS/PAGE to separate the peptide from ATP and Pi allowed to monitor simultaneously PKC autophosphorylation and phosphorylation of the peptide. The data presented here show that[Phe1499]HCV(1487-1500) can serve as a convenient tool for investigations of PKC activity also in the presence of other kinases in tissues or in crude cell extracts.     

Nickel serves as a substrate recognition motif for the endopeptidase involved in hydrogenase maturation.

The interaction of the hydrogenase maturation endopeptidase HycI with its substrate, the precursor of the large subunit, was studied. Replacement of conserved amino-acid residues in HycI, which have been shown to bind a cadmium ion from the crystallization buffer in crystals of HybD (endopeptidase for hydrogenase 2), abolished or strongly reduced processing activity. Atomic absorption spectroscopy of purified HycI and HybD proteins showed the absence of nickel. In vitro processing assays showed that the reaction requires nickel to be bound to the precursor and the protease does not have a function in nickel delivery to the substrate. Radioactive labelling of cells with 63Ni, devoid of endopeptidase, resolved several forms of the precursor which are possibly intermediates in the maturation pathway. It is concluded that the endopeptidase uses the metal in the large subunit of [NiFe]-hydrogenases as a recognition motif.     

An involucrin-like protein in hepatocytes serves as a substrate for tissue transglutaminase during apoptosis.

Cornified envelopes and apoptotic bodies are transglutaminase-cross-linked end-products of physiological cell death pathways. The two structures have similar amino acid composition. Involucrin has been considered as a cornified envelope precursor protein expressed specifically in terminally differentiating keratinocytes and squamous epithelia. We report the presence in hepatocytes of an involucrin-like protein which could be purified from dog liver with procedures characteristic to involucrins. When compared to purified dog esophagus involucrin, the liver protein also reacts with anti-involucrin antibodies, has the same relative molecular mass, possesses similar amino acid composition, and shows almost identical peptide mapping pattern. The involucrin-like protein is detectable by immunohistochemistry in normal and apoptotic hepatocytes, is a substrate of tissue transglutaminase, and is incorporated into cross-linked apoptotic bodies. These results suggest that there are overlapping molecular components in the two characteristic forms (cornification and apoptosis) of naturally occurring cell death.     

c[D-pro-Pro-D-pro-N-methyl-Ala] adopts a rigid conformation that serves as a scaffold to mimic reverse-turns

Naturally occurring cyclic tetrapeptides (CTPs) such as tentoxin (Halloin et al., Plant Physiol 1970, 45, 310-314; Saad, Phytopathology 1970, 60, 415-418), ampicidin (Darkin-Rattray, Proc Natl Acad Sci USA 1996, 93, 13143-13147), HC-toxin (Walton, Proc Natl Acad Sci USA 1987, 84, 8444-8447), and trapoxin (Yoshida and Sugita, Jpn J Cancer Res 1992, 83, 324-328; Itazaki et al., J Antibiot (Tokyo) 1990, 43, 1524-1532) have a wide range of biological activity and potential use ranging from herbicides (Walton, Proc Natl Acad Sci USA 1987, 84, 8444-8447; Judson, J Agric Food Chem 1987, 35, 451-456) to therapeutics (Loiseau, Biopolymers 2003, 69, 363-385) for malaria (Darkin-Rattray, Proc Natl Acad Sci USA 1996, 93, 13143-13147) and cancer (Yoshida and Sugita, Jpn J Cancer Res 1992, 83, 324-328). To elucidate scaffolds that have few low-energy conformations and could serve as semirigid reverse-turn mimetics, the flexibility of CTPs was determined computationally. Four analogs of cyclic tetraproline c[Pro-pro-Pro-pro] with alternating L- and D-prolines, namely c[pro-Pro-pro-NMe-Ala], c[pip-Pro-pip-Pro], c[pro-Pip-pro-Pro], and c[Ala-Pro-pip-Pro] were synthesized and characterized by NOESY NMR. Both molecular mechanics and Density Functional Theory quantum calculations found these head-to-tail CTPs to be constrained to one or two relatively stable conformations. NMR structures, while not always yielding the same lowest energy conformation as expected by in silico predictions, confirmed only one or two highly populated solution conformations for all four peptides examined. c[pro-Pro-pro-NMe-Ala] was shown to have a single all trans-amide bond conformation from both in silico predictions and NMR characterization, and to be a reverse-turn mimetic by overlapping four Calpha-Cbeta bonds with those for approximately 6.5% (Tran, J Comput Aided Mol Des 2005, 19, 551-566) of reverse-turns in the Protein Data Bank PDB with a RMSD of 0.57 A.     

The design and synthesis of a tricyclic single-nitrogen scaffold that serves as a 5-HT2C receptor agonist

5-HT2C agonists have shown efficacy in limiting food consumption and thus may serve as an important drug class in combating obesity. We describe the design and synthesis of a novel tricyclic single-nitrogen scaffold that was used to produce 5-HT2C agonists. SAR was developed around this chemotype and compounds were identified that were potent (Ki<15 nM) and selective relative to other 5-HT2 receptors. The most promising compound displayed a good pharmacokinetic profile in multiple animal species, and was efficacious in an acute feeding study in rats.     

The N-terminal domain of Rpn4 serves as a portable ubiquitin-independent degron and is recognized by specific 19S RP subunits

The number of proteasomal substrates that are degraded without prior ubiquitylation continues to grow. However, it remains poorly understood how the proteasome recognizes substrates lacking a ubiquitin (Ub) signal. Here we demonstrated that the Ub-independent degradation of Rpn4 requires the 19S regulatory particle (RP). The Ub-independent degron of Rpn4 was mapped to an N-terminal region including the first 80 residues. Inspection of its amino acid sequence revealed that the Ub-independent degron of Rpn4 consists of an intrinsically disordered domain followed by a folded segment. Using a photo-crosslinking-label transfer method, we captured three 19S RP subunits (Rpt1, Rpn2 and Rpn5) that bind the Ub-independent degron of Rpn4. This is the first time that specific 19S RP subunits have been identified interacting with a Ub-independent degron. This study provides insight into the mechanism by which Ub-independent substrates are recruited to the 26S proteasome.     

PTP-PEST, a scaffold protein tyrosine phosphatase, negatively regulates lymphocyte activation by targeting a unique set of substrates.

There is increasing interest in elucidating the mechanisms involved in the negative regulation of lymphocyte activation. Herein, we show that the cytosolic protein tyrosine phosphatase PTP-PEST is expressed abundantly in a wide variety of haemopoietic cell types, including B cells and T cells. In a model B-cell line, PTP-PEST was found to be constitutively associated with several signalling molecules, including Shc, paxillin, Csk and Cas. The interaction between Shc and PTP-PEST was augmented further by antigen receptor stimulation. Overexpression studies, antisense experiments and structure-function analyses provided evidence that PTP-PEST is an efficient negative regulator of lymphocyte activation. This function correlated with the ability of PTP-PEST to induce dephosphorylation of Shc, Pyk2, Fak and Cas, and inactivate the Ras pathway. Taken together, these data suggest that PTP-PEST is a novel and unique component of the inhibitory signalling machinery in lymphocytes.     

VIVID is a flavoprotein and serves as a fungal blue light photoreceptor for photoadaptation

Blue light regulates many physiological and developmental processes in fungi. Most of the blue light responses in the ascomycete Neurospora crassa are dependent on the two blue light regulatory proteins White Collar (WC)-1 and -2. WC-1 has recently been shown to be the first fungal blue light photoreceptor. In the present study, we characterize the Neurospora protein VIVID. VIVID shows a partial sequence similarity with plant blue light photoreceptors. In addition, we found that VIVID non-covalently binds a flavin chromophore. Upon illumination with blue light, VIVID undergoes a photocycle indicative of the formation of a flavin-cysteinyl adduct. VVD is localized in the cytoplasm and is only present after light induction. A loss-of-function vvd mutant was insensitive to increases in light intensities. Furthermore, mutational analysis of the photoactive cysteine indicated that the formation of a flavin-cysteinyl adduct is essential for VIVID functions in vivo. Our results show that VIVID is a second fungal blue light photoreceptor which enables Neurospora to perceive and respond to daily changes in light intensity.     

Phenylethanolamine is a specific substrate for type B monoamine oxidase

The characteristics of phenylethanolamine as both a competitive inhibitor and as a substrate for monoamine oxidase (MAO) were studied using rat brain and liver homogenates. Although phenylethanolamine, even at high concentrations (1 mM), produced minimal inhibition of MAO when serotonin (a substrate for type A MAO) was used as the substrate, it was a potent competitive inhibitor (K_i=11 μM) of the deamination of phenylethylamine (a substrate for type B MAO). When phenylethanolamine was used as a substrate, deprenyl, a selective inhibitor of type B MAO, was found to produce a single sigmoid inhibition curve at low concentrations of the inhibitor (pI₅₀=7.5). These results indicate that phenylethanolamine is a specific substrate for type B MAO. Identification of the products formed under the assay conditions show that phenylethanolamine is converted to both mandelic acid and phenylethylene glycol by liver homogenates but only to the latter, neutral metabolite by brain homogenates.     

Bacterially synthesized vertebrate calmodulin is a specific substrate for ubiquitination

Calmodulin purified from bacteria which express a cloned chicken calmodulin gene can be selectively conjugated with ubiquitin, using enzymes present in reticulocyte extracts. Analyses of peptide products generated from limited proteolytic digestion of the calmodulin conjugate containing a single ubiquitin indicate that lysine 115 on calmodulin is the site of linkage. This linkage site is identical to that previously reported for calmodulin purified from Dictyostelium discoideum. Substrate-dependent ATP hydrolysis by a partially purified ubiquitin conjugation enzyme system from reticulocyte extracts was used to determine the enzyme affinity to calmodulin. Km values of 7 and 9 microM were determined for dictyostelium and the bacterially expressed calmodulin, respectively. The bacterially expressed calmodulin, unlike the Dictyostelium protein, can also form conjugates containing a 2-5 molar ratio of ubiquitin but at a slower rate than that observed for conjugation at lysine 115. Results from these studies further support our hypothesis that the post-translational methylation of lysine 115 found in most forms of calmodulin serves the important function of protecting calmodulin from ubiquitination and from degradation by the cytoplasmic ubiquitin-dependent proteolytic pathway. The capability of the bacterially expressed calmodulin to form conjugates with a high molar ratio of ubiquitin suggests that the post-translational acetylation of the N terminus of calmodulin may serve a similar function.     

Paxillin serves as an ERK-regulated scaffold for coordinating FAK and Rac activation in epithelial morphogenesis

Activation of the hepatocyte growth factor (HGF) receptor in epithelial cells results in lamellipodia protrusion, spreading, migration, and tubule formation. We have previously reported that these morphogenic effects are dependent on MAPK activation at focal adhesions. In the present study we demonstrate that activated ERK phosphorylates paxillin on serine 83 and that mutation of this site eliminates HGF-stimulated increased association of paxillin and FAK in subconfluent cells. Failure to activate FAK at focal adhesions results in a loss of FAK-PI 3-kinase association and the marked reduction of Rac activation after HGF stimulation. Expression of paxillin mutants that disrupt ERK association or phosphorylation inhibits HGF-induced cell spreading, migration, and tubulogenesis. These data demonstrate that the paxillin-MAPK complex serves as a central regulator of HGF-stimulated FAK and Rac activation in the vicinity of focal adhesions, thus promoting the rapid focal adhesion turnover and lamellipodia extension that are required for migratory and tubulogenic responses.     

Analysis of cell growth kinetics and substrate diffusion in a polymer scaffold.

The cultivation of cartilage cells (chondrocytes) in polymer scaffolds leads to implants that may potentially be used to repair damaged joint cartilage or for reconstructive surgery. For this technique to be medically applicable, the physical parameters that govern cell growth in a polymer scaffold must be understood. This understanding of cell behavior under in vitro conditions, where diffusion is the primary mode of transport of nutrients, may aid in the scale-up of the cartilage generation process. A mathematical model of chondrocyte generation and nutrient consumption is developed here to analyze the behavior of cell growth in a biodegradable polymer matrix for a series of different thickness polymers. Recent literature has implied that the diffusion of nutrients is a major factor that limits cell growth (Freed et al., 1994). In the present paper, a mathematical model is developed to directly relate the effects of increasing cell mass in the polymer matrix on the transport of nutrients. Reaction and diffusion of nutrients in the cell-polymer system are described using the fundamental species continuity equations and the volume averaging method. The volume averaging method is utilized to derive a single averaged nutrient continuity equation that includes the effective transport properties. This approach allows for the derivation of effective diffusion and rate coefficients as functions of the cell volume fraction. The cell volume fraction as a function of time is determined by solution of a material balance on cell mass. Growth functions including the Moser, a modified Contois, and an nth-order heterogeneous growth kinetic model are evaluated through a parameter analysis, and the results are compared to experimental data found in the literature. The results indicate that cellular functions in conjunction with mass transfer processes can account partially for the general trends in the cell growth behavior for various thickness polymers. The Contois growth function appeared to describe the data more accurately in terms of the lag period at early times and the long time limits. However, all kinetic growth functions required variations in the kinetic parameters to fully describe the effects of polymer thickness. This result implies that restricted diffusion of nutrients is not the sole factor limiting cell growth when the thickness of the polymer is changed. Therefore, further experimental data and model improvements are needed to accurately describe the cell growth process.     

1,4-Methylhistamine is a specific substrate for type B monoamine oxidase

1,4-Methylhistamine was characterized as substrate for monoamine oxidase (MAO) in rat liver mitochondria. The $\text{K}$_m and $\text{V}$_max values were 38.8 μM and 6.33 nmoles/mg protein/60 min, respectively. The inhibition experiments with clorgyline and deprenyl, the selective inhibitors for type A and type B MAO, showed that 1,4-methylhistamine was specific for type B MAO.     

Mechanism of substrate dephosphorylation in low Mr protein tyrosine phosphatase.

Substrate dephosphorylation by the low molecular weight protein tyrosine phosphatases proceeds via nucleophilic substitution at the phosphorous atom yielding a cysteinyl phosphate intermediate. However, several questions regarding the exact reaction mechanism remain unanswered. Starting from the crystal structure of the enzyme we study the energetics of this reaction, using the empirical valence bond method in combination with molecular dynamics and free energy perturbation simulations. The free energy profiles of two mechanisms corresponding to different protonation states of the reacting groups are examined along stepwise and concerted pathways. The activation barriers calculated relative to the enzyme-substrate complex are very similar for both monoanionic and dianionic substrates, but taking the substrate binding step into account shows that hydrolysis of monoanionic substrates is strongly favored by the enzyme, because a dianionic substrate will not bind when the reacting cysteine is ionized. The calculated activation barrier for dephosphorylation of monoanionic phenyl phosphate according to this novel mechanism is 14 kcal mol(-1), which is in good agreement with experimental data. Proteins 1999;36:370-379.