Ras controls growth, survival and differentiation in the Drosophila eye by different thresholds of MAP kinase activity

Ras mediates a plethora of cellular functions during development. In the developing eye of Drosophila, Ras performs three temporally separate functions. In dividing cells, it is required for growth but is not essential for cell cycle progression. In postmitotic cells, it promotes survival and subsequent differentiation of ommatidial cells. In the present paper, we have analyzed the different roles of Ras during eye development by using molecularly defined complete and partial loss-of-function mutations of Ras. We show that the three different functions of Ras are mediated by distinct thresholds of MAPK activity. Low MAPK activity prolongs cell survival and permits differentiation of R8 photoreceptor cells while high or persistent MAPK activity is sufficient to precociously induce R1-R7 photoreceptor differentiation in dividing cells.     

pmp1+, a suppressor of calcineurin deficiency, encodes a novel MAP kinase phosphatase in fission yeast.

Calcineurin is a highly conserved and ubiquitously expressed Ca2+- and calmodulin-dependent protein phosphatase. The in vivo role of calcineurin, however, is not fully understood. Here, we show that disruption of the calcineurin gene (ppb1(+)) in fission yeast results in a drastic chloride ion (Cl-)-sensitive growth defect and that a high copy number of a novel gene pmp1(+) suppresses this defect. pmp1(+) encodes a phosphatase, most closely related to mitogen-activated protein (MAP) kinase phosphatases of the CL100/MKP-1 family. Pmp1 and calcineurin share an essential function in Cl- homeostasis, cytokinesis and cell viability. Pmp1 phosphatase dephosphorylates Pmk1, the third MAP kinase in fission yeast, in vitro and in vivo, and is bound to Pmk1 in vivo, strongly suggesting that Pmp1 negatively regulates Pmk1 MAP kinase by direct dephosphorylation. Consistently, the deletion of pmk1(+) suppresses the Cl--sensitive growth defect of ppb1 null. Thus, calcineurin and the Pmk1 MAP kinase pathway may play antagonistic functional roles in the Cl- homeostasis.     

Activation of MEK-1 and SEK-1 by Tpl-2 proto-oncoprotein, a novel MAP kinase kinase kinase.

The Tpl-2 protein serine/threonine kinase was originally identified, in a C-terminally deleted form, as the product of an oncogene associated with the progression of Moloney murine leukemia virus-induced T cell lymphomas in rats. The kinase domain of Tpl-2 is homologous to the Saccharomyces cerevisiae gene product, STE11, which encodes a MAP kinase kinase kinase. This suggested that Tpl-2 might have a similar activity. Consistent with this hypothesis, immunoprecipitated Tpl-2 and Tpl-2deltaC (a C-terminally truncated mutant) phosphorylated and activated recombinant fusion proteins of the mammalian MAP kinase kinases, MEK-1 and SEK-1, in vitro. Furthermore, transfection of Tpl-2 into COS-1 cells or Jurkat T cells. markedly activated the MAP kinases, ERK-1 and SAP kinase (JNK), which are substrates for MEK-1 and SEK-1, respectively. Tpl-2, therefore, is a MAP kinase kinase kinase which can activate two MAP kinase pathways. After Raf and Mos, Tpl-2 is the third serine/threonine oncoprotein kinase that has been shown to function as a direct activator of MEK-1.     

TMKP1 is a novel wheat stress responsive MAP kinase phosphatase localized in the nucleus

The regulation of plant signalling responses by Mitogen-Activated Protein Kinases (MAPKs)-mediated protein phosphorylation is well recognized. MAP kinase phosphatases (MKPs) are negative regulators of MAPKs in eukaryotes. We report here the identification and the characterization of TMKP1, the first wheat MKP (Triticum turgidum L. subsp. Durum). Expression profile analyses performed in two durum wheat cultivars showing a marked difference in salt and drought stress tolerance, revealed a differential regulation of TMKP1. Under salt and osmotic stress, TMKP1 is induced in the sensitive wheat variety and repressed in the tolerant one. A recombinant TMKP1 was shown to be an active phosphatase and capable to interact specifically with two wheat MAPKs (TMPK3 and TMPK6). In BY2 tobacco cells transiently expressing GFP::TMKP1, the fusion protein was localized into the nucleus. Interestingly, the deletion of the N-terminal non catalytic domain results in a strong accumulation of the truncated fusion protein in the cytoplasm. In addition, when expressed in BY2 cells, TMPK3 and TMPK6 fused to red fluorescent protein (RFP) were shown to be present predominantly in the nucleus. Surprisingly, when co-expressed with the N-terminal truncated TMKP1 fusion protein; both kinases are excluded from the nuclear compartment and accumulate in the cytoplasm. This strongly suggests that TMKP1 interacts in vivo with TMPK3 and TMPK6 and controls their subcellular localization. Taken together, our results show that the newly isolated wheat MKP might play an active role in modulating the plant cell responses to salt and osmotic stress responses.     

MKP-7, a novel mitogen-activated protein kinase phosphatase, functions as a shuttle protein

Mitogen-activated protein kinase (MAPK) phosphatases (MKPs) negatively regulate MAPK activity. In the present study, we have identified a novel MKP, designated MKP-7, and mapped it to human chromosome 12p12. MKP-7 possesses a long C-terminal stretch containing both a nuclear export signal and a nuclear localization signal, in addition to the rhodanese-like domain and the dual specificity phosphatase catalytic domain, both of which are conserved among MKP family members. When expressed in mammalian cells MKP-7 protein was localized exclusively in the cytoplasm, but this localization became exclusively nuclear following leptomycin B treatment or introduction of a mutation in the nuclear export signal. These findings indicate that MKP-7 is the first identified leptomycin B-sensitive shuttle MKP. Forced expression of MKP-7 suppressed activation of MAPKs in COS-7 cells in the order of selectivity, JNK p38 > ERK. Furthermore, a mutant form MKP-7 functioned as a dominant negative particularly against the dephosphorylation of JNK, suggesting that MKP-7 works as a JNK-specific phosphatase in vivo. Co-immunoprecipitation experiments and histological analysis suggested that MKP-7 determines the localization of MAPKs in the cytoplasm.     

Identification and characterization of a novel MAP kinase kinase kinase, MLTK

The MAPK cascades regulate a wide variety of cellular functions, including cell proliferation, differentiation, and stress responses. Here we have identified a novel MAP kinase kinase kinase (MAPKKK), termed MLTK (for MLK-like mitogen-activated protein triple kinase), whose expression is increased by activation of the ERK/MAPK pathway. There are two alternatively spliced forms of MLTK, MLTKalpha and MLTKbeta. When overexpressed in cells, both MLTKalpha and MLTKbeta are able to activate the ERK, JNK/SAPK, p38, and ERK5 pathways. Moreover, both MLTKalpha and MLTKbeta are activated in response to osmotic shock with hyperosmolar media through autophosphorylation. Remarkably, expression of MLTKalpha, but not MLTKbeta, in Swiss 3T3 cells results in the disruption of actin stress fibers and dramatic morphological changes. A kinase-dead form of MLTKalpha does not cause these phenomena. Inhibition of the p38 pathway significantly blocks MLTKalpha-induced stress fiber disruption and morphological changes. These results suggest that MLTK is a stress-activated MAPKKK that may be involved in the regulation of actin organization.     

Roles of myosin phosphatase during Drosophila development

Myosins are a superfamily of actin-dependent molecular motor proteins, among which the bipolar filament forming myosins II have been the most studied. The activity of smooth muscle/non-muscle myosin II is regulated by phosphorylation of the regulatory light chains, that in turn is modulated by the antagonistic activity of myosin light chain kinase and myosin light chain phosphatase. The phosphatase activity is mainly regulated through phosphorylation of its myosin binding subunit MYPT. To identify the function of these phosphorylation events, we have molecularly characterized the Drosophila homologue of MYPT, and analyzed its mutant phenotypes. We find that Drosophila MYPT is required for cell sheet movement during dorsal closure, morphogenesis of the eye, and ring canal growth during oogenesis. Our results indicate that the regulation of the phosphorylation of myosin regulatory light chains, or dynamic activation and inactivation of myosin II, is essential for its various functions during many developmental processes.     

PRAK, a novel protein kinase regulated by the p38 MAP kinase.

We have identified and cloned a novel serine/ threonine kinase, p38-regulated/activated protein kinase (PRAK). PRAK is a 471 amino acid protein with 20-30% sequence identity to the known MAP kinase-regulated protein kinases RSK1/2/3, MNK1/2 and MAPKAP-K2/3. PRAK was found to be expressed in all human tissues and cell lines examined. In HeLa cells, PRAK was activated in response to cellular stress and proinflammatory cytokines. PRAK activity was regulated by p38alpha and p38beta both in vitro and in vivo and Thr182 was shown to be the regulatory phosphorylation site. Activated PRAK in turn phosphorylated small heat shock protein 27 (HSP27) at the physiologically relevant sites. An in-gel kinase assay demonstrated that PRAK is a major stress-activated kinase that can phosphorylate small heat shock protein, suggesting a potential role for PRAK in mediating stress-induced HSP27 phosphorylation in vivo.     

Parcas, a regulator of non-receptor tyrosine kinase signaling, acts during anterior-posterior patterning and somatic muscle development in Drosophila melanogaster

We have isolated parcas (pcs) in a screen to identify novel regulators of muscle morphogenesis. Pcs is expressed in the ovary and oocyte during oogenesis and again in the embryo, specifically in the developing mesoderm, throughout muscle development. pcs is first required in the ovary during oogenesis for patterning and segmentation of the early Drosophila embryo due primarily to its role in the regulation of Oskar (Osk) levels. In addition to the general patterning defects observed in embryos lacking maternal contribution of pcs, these embryos show defects in Wingless (Wg) expression, causing losses of Wg-dependent cell types within the affected segment. pcs activity is required again later during embryogenesis in the developing mesoderm for muscle development. Loss and gain of function studies demonstrate that pcs is necessary at distinct times for muscle specification and morphogenesis. Pcs is predicted to be a novel regulator of non-receptor tyrosine kinase (NRTK) signaling. We have identified one target of Pcs regulation, the Drosophila Tec kinase Btk29A. While Btk29A appears to be regulated by Pcs during its early role in patterning and segmentation, it does not appear to be a major target of Pcs regulation during muscle development. We propose that Pcs fulfils its distinct roles during development by the regulation of multiple NRTKs.     

Drosophila Fascin is a novel downstream target of prostaglandin signaling during actin remodeling

Although prostaglandins (PGs)—lipid signals produced downstream of cyclooxygenase (COX) enzymes—regulate actin cytoskeletal dynamics, their mechanisms of action are unknown. We previously established Drosophila oogenesis, in particular nurse cell dumping, as a new model to determine how PGs regulate actin remodeling. PGs, and thus the Drosophila COX-like enzyme Pxt, are required for both the parallel actin filament bundle formation and the cortical actin strengthening required for dumping. Here we provide the first link between Fascin (Drosophila Singed, Sn), an actin-bundling protein, and PGs. Loss of either pxt or fascin results in similar actin defects. Fascin interacts, both pharmacologically and genetically, with PGs, as reduced Fascin levels enhance the effects of COX inhibition and synergize with reduced Pxt levels to cause both parallel bundle and cortical actin defects. Conversely, overexpression of Fascin in the germline suppresses the effects of COX inhibition and genetic loss of Pxt. These data lead to the conclusion that PGs regulate Fascin to control actin remodeling. This novel interaction has implications beyond Drosophila, as both PGs and Fascin-1, in mammalian systems, contribute to cancer cell migration and invasion.     

Expression of the receptor tyrosine kinase genes, Ror1 and Ror2, during mouse development.

In mammals, the Ror-family receptor tyrosine kinases consist of two structurally related proteins, Ror1 and Ror2, characterized by the extracellular Frizzled-like cysteine-rich domain and membrane proximal kringle domains. As an attempt to gain insights into their roles in mouse development, expression patterns of Ror1 and Ror2 during early embryogenesis were examined and compared. Interestingly, at early stages, Ror1 and Ror2 exhibit similar expression patterns in the developing face, including the frontonasal process and pharyngeal arches, which are derived from cephalic neural crest cells. On the other hand, they exhibit different expression patterns in the developing limbs and brain, where the expression of Ror2 was detected broadly compared with that of Ror1. At a later stage, both genes are expressed in a similar fashion in the developing heart and lung, yet in a distinct manner in the brain and eye.     

Identification of phosphocaveolin-1 as a novel protein tyrosine phosphatase 1B substrate

Protein tyrosine phosphatase 1B (PTP1B) is implicated in a number of signaling pathways including those mediated by insulin, epidermal growth factor (EGF), and the Src family kinases. The scaffolding protein caveolin-1 is also a participant in these pathways and is specifically phosphorylated on tyrosine 14, when these pathways are activated. Here, we provide evidence that PTP1B can efficiently catalyze the removal of the phosphoryl group from phosphocaveolin-1. Overexpression of PTP1B decreases tyrosine 14 phosphorylation in caveolin-1, while expression of the substrate-trapping mutant PTP1B/D181A causes the accumulation of phosphocaveolin-1 and prevents its dephosphorylation by endogenous PTPs. We further demonstrate that PTP1B physically associates with caveolin-1. Finally, we show that inhibition of PTP1B activity with a potent and specific small molecule PTP1B inhibitor blocks the PTP1B-catalyzed caveolin-1 dephosphorylation both in vitro and in vivo. Taken together, the results strongly suggest that caveolin-1 is a specific substrate for PTP1B. Identification of caveolin-1 as a PTP1B substrate represents an important new step in further understanding the signaling pathways regulated by PTP1B.     

Involvement of EDTP, an egg-derived tyrosine phosphatase, in the early development of Drosophila melanogaster

Previously, we purified a novel protein tyrosine phosphatase from eggs of the flesh fly, Sarcophaga peregrina. This protein tyrosine phosphatase, named egg-derived tyrosine phosphatase (EDTP), is expressed during oogenesis and early embryogenesis but is rapidly degraded in middle embryogenesis by lysosomal cathepsin L. Here, we demonstrate the requirement of EDTP in the development of the fruit fly, Drosophila melanogaster. Deletion of the Drosophila EDTP gene using transposase-catalyzed imprecise excision resulted in homozygous lethals during embryogenesis. Additionally, germline clones generated using the FLP-FRT-ovo(D) system showed severe defects in ovarian development during oogenesis. These results indicate that the Drosophila EDTP gene is crucial in oogenesis and embryogenesis.