Structural characterization of GnRH loci in the medaka genome

To help clarify the origin of a third gonadotropin-releasing hormone (GnRH) paralog found only in the teleost lineage, we have characterized GnRH loci in a teleost species, the medaka Oryzias latipes, and compared corresponding regions of the medaka and human genomes. Three GnRHs for medaka-type GnRH (mdGnRH), chicken-II-type GnRH (cGnRH-II), and salmon-type GnRH (sGnRH) exist as single-copy genes and reside on separate chromosomes in the medaka genome. Both medaka mdGnRH and human mGnRH are closely linked to FLJ20038 encoding a hypothetical protein, and both cGnRH-IIs in the medaka and humans are adjacent to PTP(alpha) for protein tyrosine phosphatase alpha. These conserved syntenies demonstrate that mdGnRH and cGnRH-II in teleosts are orthologous to mGnRH and cGnRH-II in tetrapods, respectively. On the other hand, the third paralogous GnRH in the medaka, sGnRH, is adjacent to PTP(epsilon), a paralog of PTP(alpha). Although humans possess PTP(epsilon) on 10q26, no sGnRH-like sequence was found in the human genome databases. Therefore a gene duplication that gave rise to the third paralogous GnRH likely occurred before the divergence of teleosts and tetrapods, and it has been lost only in the tetrapod lineage. Additionally, together with the prior observations that like GnRH, PTP(alpha)/PTP(epsilon) are strongly expressed in neural and tumor cells and that GnRH can increase PTP activity, the current data suggests that the physically linked cGnRH-II/sGnRH and PTP(alpha)/PTP(epsilon) are also functionally linked.     

Residue 259 is a key determinant of substrate specificity of protein-tyrosine phosphatases 1B and alpha

The aim of this study was to define the structural elements that determine the differences in substrate recognition capacity of two protein-tyrosine phosphatases (PTPs), PTP1B and PTPalpha, both suggested to be negative regulators of insulin signaling. Since the Ac-DADE(pY)L-NH(2) peptide is well recognized by PTP1B, but less efficiently by PTPalpha, it was chosen as a tool for these analyses. Calpha regiovariation analyses and primary sequence alignments indicate that residues 47, 48, 258, and 259 (PTP1B numbering) define a selectivity-determining region. By analyzing a set of DADE(pY)L analogs with a series of PTP mutants in which these four residues were exchanged between PTP1B and PTPalpha, either in combination or alone, we here demonstrate that the key selectivity-determining residue is 259. In PTPalpha, this residue is a glutamine causing steric hindrance and in PTP1B a glycine allowing broad substrate recognition. Significantly, replacing Gln(259) with a glycine almost turns PTPalpha into a PTP1B-like enzyme. By using a novel set of PTP inhibitors and x-ray crystallography, we further provide evidence that Gln(259) in PTPalpha plays a dual role leading to restricted substrate recognition (directly via steric hindrance) and reduced catalytic activity (indirectly via Gln(262)). Both effects may indicate that PTPalpha regulates highly selective signal transduction processes.     

Integrin-induced tyrosine phosphorylation of protein-tyrosine phosphatase-alpha is required for cytoskeletal reorganization and cell migration

Protein-tyrosine phosphatase-alpha (PTPalpha) activates Src family kinases (SFKs) to promote the integrin-stimulated early autophosphorylation of focal adhesion kinase (FAK). We report here that integrin stimulation induces tyrosine phosphorylation of PTPalpha. PTPalpha was dephosphorylated upon fibroblast detachment from the substratum and rephosphorylated when cells were plated on the integrin ligand fibronectin. alpha PTP phosphorylation occurred at Tyr789 and required SFKs (Src or Fyn/Yes), FAK, and an intact cytoskeleton. It also required active PTPalpha or constitutively active Src. These observations indicate that PTPalpha activates SFKs and that the subsequently activated SFK.FAK tyrosine kinase complex in turn phosphorylates PTPalpha. Reintroduction of wild-type PTPalpha or unphosphorylatable PTPalpha(Y789F) (but not inactive PTPalpha) into PTPalpha-null fibroblasts restored defective integrin-induced SFK activation, FAK phosphorylation, and paxillin phosphorylation. PTPalpha(Y789F) and inactive PTPalpha could not rescue delayed actin stress fiber assembly and focal adhesion formation or defective cell migration. This study distinguishes two roles of PTPalpha in integrin signaling: an early role as an activator of SFKs and FAK with no requirement for PTPalpha phosphorylation and a later downstream role in cytoskeleton-associated events for which PTPalpha phosphorylation at Tyr789 is essential.     

Characterization on the alternative splicing, expression and gene phylogenesis of PTPR4 family in Japanese flounder, Paralichthys olivaceus

One mechanism of eukaryotic signaling is protein phosphorylation by protein tyrosine phosphatases (PTPs). Here we have identified the PTP Receptor-Type IV (PTPR4) family, including one form of PTPalpha and two forms of PTPepsilon (PTPepsilon M and PTPepsilon C) in flounder. The existence of PTPepsilon C has not been reported in non-mammalian animals. Semi-quantitative RT-PCR revealed independent expression patterns and levels of PTPalpha and the two forms of PTPepsilon in various tissues. The sequence of PTPepsilon C was identical to that of PTPepsilon M except for its 5'-terminal regions. Southern blot analysis proved that there existed only one PTPepsilon gene in flounder genome, indicating that the two isoforms of PTPepsilon might have been derived from alternative splicing of the single gene. Phylogenetic analysis of PTP domain D2 and part of D1 of PTPR4 showed that flounder was first joint with other teleost fish and then tetrapods, and also provided evidence that the gene duplication from the ancestor gene to PTPalpha and PTPepsilon occurred before the divergence of Gnathastomata and Agnatha. These results showed that the functional evolution of protein phosphorylation is promoted by not only genome duplication, but also elaborate regulation of gene expression.     

A novel form of gonadotropin-releasing hormone in the medaka, Oryzias latipes

The present study has identified three molecular forms of gonadotropin-releasing hormone (GnRH) in the brain of a teleost, the medaka, by isolation of their cDNAs. This species has a novel GnRH, which is here named medaka-type GnRH (mdGnRH), in addition to two characterized forms, chicken-II-type GnRH (cGnRH-II) and salmon-type GnRH (sGnRH). Phylogenetic analysis showed that mdGnRH is a medaka homolog of and seabream-type GnRH (sbGnRH) and mammalian-type GnRH (mGnRH) in other species, and suggested that all vertebrates have three distinct GnRHs. Furthermore, in situ hybridization revealed that the mdGnRH gene is expressed only in neurons clustered within the preoptic area as sbGnRH and mGnRH genes in other species are, while the genes for cGnRH-II and sGnRH are only in the midbrain tegmentum and nucleus olfactoretinalis, respectively. This result suggested that mdGnRH is a hypophysiotropic factor and the other two forms are involved in other physiological events as neuromodulators or neurotransmitters.     

Differential function of PTPalpha and PTPalpha Y789F in T cells and regulation of PTPalpha phosphorylation at Tyr-789 by CD45

CD45 is a major membrane protein tyrosine phosphatase (PTP) expressed in T cells where it regulates the activity of Lck, a Src family kinase important for T cell receptor-mediated activation. PTPalpha is a more widely expressed transmembrane PTP that has been shown to regulate the Src family kinases, Src and Fyn, and is also present in T cells. Here, PTPalpha was phosphorylated at Tyr-789 in CD45(-) T cells but not in CD45(+) T cells suggesting that CD45 could regulate the phosphorylation of PTPalpha at this site. Furthermore, CD45 could directly dephosphorylate PTPalpha in vitro. Expression of PTPalpha and PTPalpha-Y789F in T cells revealed that the mutant had a reduced ability to decrease Fyn and Cbp phosphorylation, to regulate the kinase activity of Fyn, and to restore T cell receptor-induced signaling events when compared with PTPalpha. Conversely, this mutant had an increased ability to prevent Pyk2 phosphorylation and CD44-mediated cell spreading when compared with PTPalpha. These data demonstrate distinct activities of PTPalpha and PTPalpha-Y789F in T cells and identify CD45 as a regulator of PTPalpha phosphorylation at tyrosine 789 in T cells.     

Suramin derivatives as inhibitors and activators of protein-tyrosine phosphatases

Protein-tyrosine phosphatases (PTPs) are important signaling enzymes that have emerged within the last decade as a new class of drug targets. It has previously been shown that suramin is a potent, reversible, and competitive inhibitor of PTP1B and Yersinia PTP (YopH). We therefore screened 45 suramin analogs against a panel of seven PTPs, including PTP1B, YopH, CD45, Cdc25A, VHR, PTPalpha, and LAR, to identify compounds with improved potency and specificity. Of the 45 compounds, we found 11 to have inhibitory potency comparable or significantly improved relative to suramin. We also found suramin to be a potent inhibitor (IC(50) = 1.5 microm) of Cdc25A, a phosphatase that mediates cell cycle progression and a potential target for cancer therapy. In addition we also found three other compounds, NF201, NF336, and NF339, to be potent (IC(50) < 5 microm) and specific (at least 20-30-fold specificity with respect to the other human PTPs tested) inhibitors of Cdc25A. Significantly, we found two potent and specific inhibitors, NF250 and NF290, for YopH, the phosphatase that is an essential virulence factor for bubonic plague. Two of the compounds tested, NF504 and NF506, had significantly improved potency as PTP inhibitors for all phosphatases tested except for LAR and PTPalpha. Surprisingly, we found that a significant number of these compounds activated the receptor-like phosphatases, PTPalpha and LAR. In further characterizing this activation phenomenon, we reveal a novel role for the membrane-distal cytoplasmic PTP domain (D2) of PTPalpha: the direct intramolecular regulation of the activity of the membrane-proximal cytoplasmic PTP domain (D1). Binding of certain of these compounds to PTPalpha disrupts D1-D2 basal state contacts and allows new contacts to occur between D1 and D2, which activates D1 by as much as 12-14-fold when these contacts are optimized.     

Protein tyrosine phosphatase alpha regulates Fyn activity and Cbp/PAG phosphorylation in thymocyte lipid rafts

A role for the receptor protein tyrosine phosphatase alpha (PTPalpha) in immune cell function and regulation of Src family kinases was investigated using thymocytes from PTPalpha-deficient mice. PTPalpha-null thymocytes develop normally, but unstimulated PTPalpha-/- cells exhibit increased tyrosine phosphorylation of specific proteins, increased Fyn activity, and hyperphosphorylation of Cbp/PAG that promotes its association with C-terminal Src kinase. Elevated Fyn activity in the absence of PTPalpha is due to enhanced phosphorylation of Fyn tyrosines 528 and 417. Some PTPalpha is localized in lipid rafts of thymocytes, and raft-associated Fyn is specifically activated in PTPalpha-/- cells. PTPalpha is not a Cbp/PAG phosphatase, because it is not required for Cbp/PAG dephosphorylation in unstimulated or anti-CD3-stimulated thymocytes. Together, our results indicate that PTPalpha, likely located in lipid rafts, regulates the activity of raft Fyn. In the absence of PTPalpha this population of Fyn is activated and phosphorylates Cbp/PAG to enhance association with C-terminal Src kinase. Although TCR-mediated tyrosine phosphorylation was apparently unaffected by the absence of PTPalpha, the long-term proliferative response of PTPalpha-/- thymocytes was reduced. These findings indicate that PTPalpha is a component of the complex Src family tyrosine kinase regulatory network in thymocytes and is required to suppress Fyn activity in unstimulated cells in a manner that is not compensated for by the major T cell PTP and SFK regulator, CD45.     

Tyrosine phosphatase PTPalpha regulates focal adhesion remodeling through Rac1 activation

We characterized the role of protein tyrosine phosphatase (PTP)-alpha in focal adhesion (FA) formation and remodeling using wild-type and PTPalpha-deficient (PTPalpha(-/-)) cells. Compared with wild-type cells, spreading PTPalpha(-/-) fibroblasts displayed fewer leading edges and formed elongated alpha-actinin-enriched FA at the cell periphery. These features suggest the presence of slowly remodeling cell adhesions and were phenocopied in human fibroblasts in which PTPalpha was knocked down using short interfering RNA (siRNA) or in NIH-3T3 fibroblasts expressing catalytically inactive (C433S/C723S) PTPalpha. Fluorescence recovery after photobleaching showed slower green fluorescence protein-alpha-actinin recovery in the FA of PTPalpha(-/-) than wild-type cells. These alterations correlated with reduced cell spreading, adhesion, and polarization and retarded contraction of extracellular matrices in PTPalpha(-/-) fibroblasts. Activation of Rac1 and its recruitment to FA during spreading were diminished in cells expressing C433S/C723S PTPalpha. Rac1(-/-) cells also displayed abnormally elongated and peripherally distributed FA that failed to remodel. Conversely, expression of constitutively active Rac1 restored normal FA remodeling in PTPalpha(-/-) cells. We conclude that PTPalpha is required for remodeling of FA during cell spreading via a pathway involving Rac1.     

Tyrosine phosphatases epsilon and alpha perform specific and overlapping functions in regulation of voltage-gated potassium channels in Schwann cells

Tyrosine phosphatases (PTPs) epsilon and alpha are closely related and share several molecular functions, such as regulation of Src family kinases and voltage-gated potassium (Kv) channels. Functional interrelationships between PTPepsilon and PTPalpha and the mechanisms by which they regulate K+ channels and Src were analyzed in vivo in mice lacking either or both PTPs. Lack of either PTP increases Kv channel activity and phosphorylation in Schwann cells, indicating these PTPs inhibit Kv current amplitude in vivo. Open probability and unitary conductance of Kv channels are unchanged, suggesting an effect on channel number or organization. PTPalpha inhibits Kv channels more strongly than PTPepsilon; this correlates with constitutive association of PTPalpha with Kv2.1, driven by membranal localization of PTPalpha. PTPalpha, but not PTPepsilon, activates Src in sciatic nerve extracts, suggesting Src deregulation is not responsible exclusively for the observed phenotypes and highlighting an unexpected difference between both PTPs. Developmentally, sciatic nerve myelination is reduced transiently in mice lacking either PTP and more so in mice lacking both PTPs, suggesting both PTPs support myelination but are not fully redundant. We conclude that PTPepsilon and PTPalpha differ significantly in their regulation of Kv channels and Src in the system examined and that similarity between PTPs does not necessarily result in full functional redundancy in vivo.     

Mitotic activation of protein-tyrosine phosphatase alpha and regulation of its Src-mediated transforming activity by its sites of protein kinase C phosphorylation

During mitosis, the catalytic activity of protein-tyrosine phosphatase (PTP) alpha is enhanced, and its inhibitory binding to Grb2, which specifically blocks Src dephosphorylation, is decreased. These effects act synergistically to activate Src in mitosis. We show here that these effects are abrogated by mutation of Ser180 and/or Ser204, the sites of protein kinase C-mediated phosphorylation within PTPalpha. Moreover, either a Ser-to-Ala substitution or serine dephosphorylation specifically eliminated the ability of PTPalpha to dephosphorylate and activate Src even during interphase. This explains why the substitutions eliminated PTPalpha transforming activity, even though PTPalpha interphase dephosphorylation of nonspecific substrates was only slightly decreased. This occurred without change in the phosphorylation of PTPalpha at Tyr789, which is required for "phosphotyrosine displacement" during Src dephosphorylation. Thus, in addition to increasing PTPalpha nonspecific catalytic activity, Ser180 and Ser204 phosphorylation (along with Tyr789 phosphorylation) regulates PTPalpha substrate specificity. This involves serine phosphorylation-dependent differential modulation of the affinity of Tyr(P)789 for the Src and Grb2 SH2 domains. The results suggest that protein kinase C may participate in the mitotic activation of PTPalpha and Src and that there are intramolecular interactions between the PTPalpha C-terminal and membrane-proximal regions that are regulated, at least in part, by serine phosphorylation.     

Protein tyrosine phosphatase alpha (PTPalpha) and contactin form a novel neuronal receptor complex linked to the intracellular tyrosine kinase fyn

Glycosyl phosphatidylinositol (GPI)-linked receptors and receptor protein tyrosine phosphatases (RPTPs), both play key roles in nervous system development, although the molecular mechanisms are largely unknown. Despite lacking a transmembrane domain, GPI receptors can recruit intracellular src family tyrosine kinases to receptor complexes. Few ligands for the extracellular regions of RPTPs are known, relegating most to the status of orphan receptors. We demonstrate that PTPalpha, an RPTP that dephosphorylates and activates src family kinases, forms a novel membrane-spanning complex with the neuronal GPI-anchored receptor contactin. PTPalpha and contactin associate in a lateral (cis) complex mediated through the extracellular region of PTPalpha. This complex is stable to isolation from brain lysates or transfected cells through immunoprecipitation and to antibody-induced coclustering of PTPalpha and contactin within cells. This is the first demonstration of a receptor PTP in a cis configuration with another cell surface receptor, suggesting an additional mode for regulation of a PTP. The transmembrane and catalytic nature of PTPalpha indicate that it likely forms the transducing element of the complex, and we postulate that the role of contactin is to assemble a phosphorylation-competent system at the cell surface, conferring a dynamic signal transduction capability to the recognition element.     

Residue 259 in protein-tyrosine phosphatase PTP1B and PTPalpha determines the flexibility of glutamine 262

To study the flexibility of the substrate-binding site and in particular of Gln262, we have performed adiabatic conformational search and molecular dynamics simulations on the crystal structure of the catalytic domain of wild-type protein-tyrosine phosphatase (PTP) 1B, a mutant PTP1B(R47V,D48N,M258C,G259Q), and a model of the catalytically active form of PTPalpha. For each molecule two cases were modeled: the Michaelis-Menten complex with the substrate analogue p-nitrophenyl phosphate (p-PNPP) bound to the active site and the cysteine-phosphor complex, each corresponding to the first and second step of the phosphate hydrolysis. Analyses of the trajectories revealed that in the cysteine-phosphor complex of PTP1B, Gln262 oscillates freely between the bound phosphate group and Gly259 frequently forming, as observed in the crystal structure, a hydrogen bond with the backbone oxygen of Gly259. In contrast, the movement of Gln262 is restricted in PTPalpha and the mutant due to interactions with Gln259 reducing the frequency of the oscillation of Gln262 and thereby delaying the positioning of this residue for the second step in the catalysis, as reflected experimentally by a reduction in k(cat). Additionally, in the simulation with the Michaelis-Menten complexes, we found that a glutamine in position 259 induces steric hindrance by pushing the Gln262 side chain further toward the substrate and thereby negatively affecting K(m) as indicated by kinetic studies. Detailed analysis of the water structure around Gln262 and the active site Cys215 reveals that the probability of finding a water molecule correctly positioned for catalysis is much larger in PTP1B than in PTP1B(R47V,D48N,M258C,G259Q) and PTPalpha, in accordance with experiments.     

Enzyme kinetic characterization of protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) play a central role in cellular signaling processes, resulting in an increased interest in modulating the activities of PTPs. We therefore decided to undertake a detailed enzyme kinetic evaluation of various transmembrane and cytosolic PTPs (PTPalpha, PTPbeta, PTPepsilon, CD45, LAR, PTP1B and SHP-1), using pNPP as substrate. Most noticeable is the increase in the turnover number for PTPbeta with increasing pH and the weak pH-dependence of the turnover number of CD45. The kinetic data for PTPalpha-D1 and PTPalpha-D1D2 suggest that D2 affects the catalysis of pNPP. PTPepsilon and the closely homologous PTPalpha behave differently. The K(m) data were lower for PTPepsilon than those for PTPalpha, while the inverse was observed for the catalytic efficiencies.     

Potent and highly selective inhibitors of the protein tyrosine phosphatase 1B

Several protein tyrosine phosphatases (PTPases) have been implicated as regulatory agents in the insulin-stimulated signal transduction pathway, including PTP1B, PTPalpha, and LAR. Furthermore, since all three enzymes are suggested to serve as negative regulators of insulin signaling, one or more may play a pivotal role in the pathogenesis of insulin resistance. We report herein the acquisition of highly selective PTP1B-targeted inhibitors. We recently demonstrated that PTP1B contains two proximal aromatic phosphate binding sites [Puius, Y. A., Zhao, Y., Sullivan, M., Lawrence, D. S., Almo S. C., and Zhang, Z. Y. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 13420-5], and we have now employed this structural feature to design and synthesize an array of bis(aryldifluorophosphonates). Not only do the lead compounds serve as potent inhibitors of PTP1B but, in addition, several exhibit selectivities for PTP1B versus PTPalpha, LAR, and VHR that are greater than 2 orders in magnitude.     

Effects of kisspeptin1 on electrical activity of an extrahypothalamic population of gonadotropin-releasing hormone neurons in medaka (Oryzias latipes)

Kisspeptin (product of the kiss1 gene) is the most potent known activator of the hypothalamo-pituitary-gonadal axis. Both kiss1 and the kisspeptin receptor are highly expressed in the hypothalamus of vertebrates, and low doses of kisspeptin have a robust and long-lasting stimulatory effect on the rate of action potential firing of hypophysiotropic gonadotropin releasing hormone-1 (GnRH1) neurons in mice. Fish have multiple populations of GnRH neurons distinguished by their location in the brain and the GnRH gene that they express. GnRH3 neurons located in the terminal nerve (TN) associated with the olfactory bulb are neuromodulatory and do not play a direct role in regulating pituitary-gonadal function. In medaka fish, the electrical activity of TN-GnRH3 neurons is modulated by visual cues from conspecifics, and is thought to act as a transmitter of information from the external environment to the central nervous system. TN-GnRH3 neurons also play a role in sexual motivation and arousal states, making them an important population of neurons to study for understanding coordination of complex behaviors. We investigated the role of kisspeptin in regulating electrical activity of TN-GnRH3 neurons in adult medaka. Using electrophysiology in an intact brain preparation, we show that a relatively brief treatment with 100 nM of kisspeptin had a long-lasting stimulatory effect on the electrical activity of an extrahypothalamic population of GnRH neurons. Dose-response analysis suggests a relatively narrow activational range of this neuropeptide. Further, blocking action potential firing with tetrodotoxin and blocking synaptic transmission with a low Ca(2+)/high Mg(2+) solution inhibited the stimulatory action of kisspeptin on electrical activity, indicating that kisspeptin is acting indirectly through synaptic regulation to excite TN-GnRH3 neurons. Our findings provide a new perspective on kisspeptin's broader functions within the central nervous system, through its regulation of an extrahypothalamic population of GnRH neurons involved in multiple neuromodulatory functions.     

Characterization of Hox genes in the bichir,Polypterus palmas

It has been suggested that the increase in the number of Hox genes may have been one of the key events in vertebrate evolution. Invertebrates have one Hox cluster, while mammals have four. Interestingly, the number of Hox gene clusters is greater in the teleost fishes, zebrafish and medaka, than in mouse and human. The greater number of Hox clusters in the teleosts suggests that Hox gene duplication events have occurred during the radiation of ray-finned fishes. The question is when the Hox gene duplication event(s) that lead to seven Hox clusters in the teleosts actually occurred.We have addressed this question by studying the Hox genes in the bichir, Polypterus palmas. A preliminary PCR-estimation of the number of Hox genes suggests that Polypterus has five different Hox9 cognate group genes, which may be an indication of more than four Hox clusters in the bichir.