Understanding Pseudophosphatase Function Through Biochemical Interactions.

Pseudophosphatases have been solidified as important signaling molecules that regulate signal transduction cascades. However, their mechanisms of action remain enigmatic. Reflecting this mystery, the prototypical pseudophosphatase STYX (phospho-serine-threonine/tyrosine-binding protein) was named with allusion to the river of the dead in Greek mythology to emphasize that these molecules are "dead" phosphatases. Although proteins with STYX domains do not catalyze dephosphorylation, this does not preclude their having other functions, including as integral elements of signaling networks. Thus, understanding their roles may mark them as potential novel drug targets. This chapter outlines common strategies used to characterize the functions of pseudophosphatases, using as an example MK-STYX [MAPK (mitogen-activated protein kinase) phospho-serine-threonine/tyrosine-binding], which has been linked to tumorigenesis, hepatocellular carcinoma, glioblastoma, apoptosis, and neuronal differentiation. We start with the importance of "restoring" (when possible) phosphatase activity in a pseudophosphatase, so the active mutant may be used as a comparison control throughout immunoprecipitation and mass spectrometry analyses. To this end, we provide protocols for site-directed mutagenesis, mammalian cell transfection, co-immunoprecipitation, phosphatase activity assays, and immunoblotting that we have used to investigate MK-STYX and the active mutant MK-STYXactive. We also highlight the importance of utilizing RNA interference (RNAi) "knockdown" technology to determine a cellular phenotype in various cell lines. Therefore, we outline our protocols for introducing short hairpin RNA (shRNA) expression plasmids into mammalian cells and quantifying knockdown of gene expression with real-time quantitative PCR (qPCR). We also provide a bioinformatic approach to investigating MK-STYX and MK-STYX(active mutant). These bioinformatic approaches can stand alone experimentally but also complement and enhance "wet" bench approaches such as binding assays and/or activity assays. A combination of cellular, molecular, biochemical, proteomic, and bioinformatic techniques has been a powerful tool in identifying novel functions of MK-STYX. Likewise, the information provided here should be a helpful guide to elucidating the functions of other pseudophosphatases.

The DUSP domain of pseudophosphatase MK-STYX interacts with G3BP1 to decrease stress granules.

Mitogen activated protein kinase phosphoserine/threonine/tyrosine-binding protein (MK-STYX) is a dual specificity (DUSP) member of the protein tyrosine phosphatase family. It is a pseudophosphatase, which lacks the essential amino acids histidine and cysteine in the catalytic active signature motif (HCX5R). We previously reported that MK-STYX interacts with G3BP1 [Ras-GAP (GTPase-activating protein) SH3 (Src homology 3) domain-binding-1] and reduces stress granules, stalled mRNA. To determine how MK-STYX reduces stress granules, truncated domains, CH2 (cell division cycle 25 phosphatase homology 2) and DUSP, of MK-STYX were used. Wild-type MK-STYX and the DUSP domain significantly decreased stressed granules that were induced by sodium arsenite, in which G3BP1 (a stress granule nucleator) was used as the marker. In addition, HEK/293 and HeLa cells co-expressing G3BP1-GFP and mCherry-MK-STYX, mCherry-MK-STYX-CH2, mCherry-MK-STYX-DUSP or mCherry showed that stress granules were significantly decreased in the presence of wild-type MK-STYX and the DUSP domain of MK-STYX. Further characterization of these dynamics in HeLa cells showed that the CH2 domain increased the number of stress granules within a cell, relative to wild-type and DUSP domain of MK-STYX. To further analyze the interaction of G3BP1 and the domains of MK-STYX, coimmunoprecipitation experiments were performed. Cells co-expressing G3BP1-GFP and mCherry, mCherry-MK-STYX, mCherry-MK-STYX-CH2, or mCherry-MK-STYX-DUSP demonstrated that the DUSP domain of MK-STYX interacts with both G3BP1-GFP and endogenous G3BP1, whereas the CH2 domain of MK-STYX did not coimmunoprecipitate with G3BP1. In addition, G3BP1 tyrosine phosphorylation, which is required for stress granule formation, was decreased in the presence of wild-type MK-STYX or the DUSP domain but increased in the presence of CH2. These data highlight a model for how MK-STYX decreases G3BP1-induced stress granules. The DUSP domain of MK-STYX interacts with G3BP1 and negatively alters its tyrosine phosphorylation- decreasing stress granule formation.

Evolutionary genomic relationships and coupling in MK-STYX and STYX pseudophosphatases.

The dual specificity phosphatase (DUSP) family has catalytically inactive members, called pseudophosphatases. They have mutations in their catalytic motifs that render them enzymatically inactive. This study analyzes the significance of two pseudophosphatases, MK-STYX [MAPK (mitogen-activated protein kinase phosphoserine/threonine/tyrosine-binding protein]) and STYX (serine/threonine/tyrosine-interacting protein), throughout their evolution and provides measurements and comparison of their evolutionary conservation. Phylogenetic trees were constructed to show any deviation from various species evolutionary paths. Data was collected on a large set of proteins that have either one of the two domains of MK-STYX, the DUSP domain or the cdc-25 homology (CH2) /rhodanese-like domain. The distance between species pairs for MK-STYX or STYX and Ka/Ks ratio were calculated. In addition, both pseudophosphatases were ranked among a large set of related proteins, including the active homologs of MK-STYX, MKP (MAPK phosphatase)-1 and MKP-3. MK-STYX had one of the highest species-species protein distances and was under weaker purifying selection pressure than most proteins with its domains. In contrast, the protein distances of STYX were lower than 82% of the DUSP-containing proteins and was under one of the strongest purifying selection pressures. However, there was similar selection pressure on the N-terminal sequences of MK-STYX, STYX, MKP-1, and MKP-3. We next perform statistical coupling analysis, a process that reveals interconnected regions within the proteins. We find that while MKP-1,-3, and STYX all have 2 functional units (sectors), MK-STYX only has one, and that MK-STYX is similar to MKP-3 in the evolutionary coupling of the active site and KIM domain. Within those two domains, the mean coupling is also most similar for MK-STYX and MKP-3. This study reveals striking distinctions between the evolutionary patterns of MK-STYX and STYX, suggesting a very specific role for each pseudophosphatase, further highlighting the relevance of these atypical members of DUSP as signaling regulators. Therefore, our study provides computational evidence and evolutionary reasons to further explore the properties of pseudophosphatases, in particular MK-STYX and STYX.

Pseudophosphatases as Regulators of MAPK Signaling.

Mitogen-activated protein kinase (MAPK) signaling pathways are highly conserved regulators of eukaryotic cell function. These enzymes regulate many biological processes, including the cell cycle, apoptosis, differentiation, protein biosynthesis, and oncogenesis; therefore, tight control of the activity of MAPK is critical. Kinases and phosphatases are well established as MAPK activators and inhibitors, respectively. Kinases phosphorylate MAPKs, initiating and controlling the amplitude of the activation. In contrast, MAPK phosphatases (MKPs) dephosphorylate MAPKs, downregulating and controlling the duration of the signal. In addition, within the past decade, pseudoenzymes of these two families, pseudokinases and pseudophosphatases, have emerged as bona fide signaling regulators. This review discusses the role of pseudophosphatases in MAPK signaling, highlighting the function of phosphoserine/threonine/tyrosine-interacting protein (STYX) and TAK1-binding protein (TAB 1) in regulating MAPKs. Finally, a new paradigm is considered for this well-studied cellular pathway, and signal transduction pathways in general.

The Roles of Pseudophosphatases in Disease.

The pseudophosphatases, atypical members of the protein tyrosine phosphatase family, have emerged as bona fide signaling regulators within the past two decades. Their roles as regulators have led to a renaissance of the pseudophosphatase and pseudoenyme fields, catapulting interest from a mere curiosity to intriguing and relevant proteins to investigate. Pseudophosphatases make up approximately fourteen percent of the phosphatase family, and are conserved throughout evolution. Pseudophosphatases, along with pseudokinases, are important players in physiology and pathophysiology. These atypical members of the protein tyrosine phosphatase and protein tyrosine kinase superfamily, respectively, are rendered catalytically inactive through mutations within their catalytic active signature motif and/or other important domains required for catalysis. This new interest in the pursuit of the relevant functions of these proteins has resulted in an elucidation of their roles in signaling cascades and diseases. There is a rapid accumulation of knowledge of diseases linked to their dysregulation, such as neuropathies and various cancers. This review analyzes the involvement of pseudophosphatases in diseases, highlighting the function of various role(s) of pseudophosphatases involvement in pathologies, and thus providing a platform to strongly consider them as key therapeutic drug targets.

Pseudophosphatase MK-STYX: the atypical member of the MAP kinase phosphatases.

The regulation of the phosphorylation of mitogen-activated protein kinases (MAPKs) is essential for cellular processes such as proliferation, differentiation, survival, and death. Mutations within the MAPK signaling cascades are implicated in diseases such as cancer, neurodegenerative disorders, arthritis, obesity, and diabetes. MAPK phosphorylation is controlled by an intricate balance between MAPK kinases (enzymes that add phosphate groups) and MAPK phosphatases (MKPs) (enzymes that remove phosphate groups). MKPs are complex negative regulators of the MAPK pathway that control the amplitude and spatiotemporal regulation of MAPKs. MK-STYX (MAPK phosphoserine/threonine/tyrosine-binding protein) is a member of the MKP subfamily, which lacks the critical histidine and nucleophilic cysteine residues in the active site required for catalysis. MK-STYX does not influence the phosphorylation status of MAPK, but even so it adds to the complexity of signal transduction cascades as a signaling regulator. This review highlights the function of MK-STYX, providing insight into MK-STYX as a signal regulating molecule in the stress response, HDAC 6 dynamics, apoptosis, and neurite differentiation.

Mislocalization of Cancer-associated Thyroid Hormone Receptor Mutants.

The thyroid hormone receptor (TR) is essential for the proper regulation of metabolism and development, as it regulates gene expression in response to thyroid hormone. Nuclear localization signals (NLSs) and nuclear export signals (NESs) allow for TR transport into and out of the nucleus, respectively. Previous research suggests that nuclear import, nuclear retention, and nuclear export of TR are associated with modulation of gene expression, the alteration of which can contribute to various diseases. Here, we examined the impact of cancer-associated mutations on TR localization patterns as a way of analyzing key structural components of TR and to further explore the correlation between TR trafficking, misfolding, and disease. Through mammalian cell transfection of expression plasmids for green fluorescent protein (GFP) and mCherry-tagged TRα1 and quantitative fluorescence microscopy, we examined particular groups of TRα1 mutations that were observed in patients with hepatocellular carcinoma, renal cell carcinoma, and thyroid cancer, and are associated with NLSs and NESs of TRα1. We also investigated structural alterations of the mutants by in silico modeling. Our results show striking shifts towards a more cytoplasmic localization for many of the mutants and an increased tendency to form cytosolic and nuclear aggregates.

Pseudophosphatase MK-STYX Alters Histone Deacetylase 6 Cytoplasmic Localization, Decreases Its Phosphorylation, and Increases Detyrosination of Tubulin.

The catalytically inactive mitogen-activated protein (MAP) kinase phosphatase, MK-STYX (MAPK (mitogen-activated protein kinase) phosphoserine/threonine/tyrosine-binding protein) interacts with the stress granule nucleator G3BP-1 (Ras-GAP (GTPase-activating protein) SH3 (Src homology 3) domain-binding protein-1), and decreases stress granule (stalled mRNA) formation. Histone deacetylase isoform 6 (HDAC6) also binds G3BP-1 and serves as a major component of stress granules. The discovery that MK-STYX and HDAC6 both interact with G3BP-1 led us to investigate the effects of MK-STYX on HDAC6 dynamics. In control HEK/293 cells, HDAC6 was cytosolic, as expected, and formed aggregates under conditions of stress. In contrast, in cells overexpressing MK-STYX, HDAC6 was both nuclear and cytosolic and the number of stress-induced aggregates significantly decreased. Immunoblots showed that MK-STYX decreases HDAC6 serine phosphorylation, protein tyrosine phosphorylation, and lysine acetylation. HDAC6 is known to regulate microtubule dynamics to form aggregates. MK-STYX did not affect the organization of microtubules, but did affect their post-translational modification. Tubulin acetylation was increased in the presence of MK-STYX. In addition, the detyrosination of tubulin was significantly increased in the presence of MK-STYX. These findings show that MK-STYX decreases the number of HDAC6-containing aggregates and alters their localization, sustains microtubule acetylation, and increases detyrosination of microtubules, implicating MK-STYX as a signaling molecule in HDAC6 activity.

The role of pseudophosphatases as signaling regulators.

Pseudophosphatases are atypical members of the protein tyrosine phosphatase superfamily. Mutations within their catalytic signature motif render them catalytically inactive. Despite this lack of catalytic function, pseudophosphatases have been implicated in various diseases such as Charcot Marie-Tooth disorder, cancer, metabolic disorder, and obesity. Moreover, they have roles in various signaling networks such as spermatogenesis, apoptosis, stress response, tumorigenesis, and neurite differentiation. This review highlights the roles of pseudophosphatases as essential regulators in signaling cascades, providing insight into the function of these catalytically inactive enzymes.

MK-STYX Alters the Morphology of Primary Neurons, and Outgrowths in MK-STYX Overexpressing PC-12 Cells Develop a Neuronal Phenotype.

We previously reported that the pseudophosphatase MK-STYX (mitogen activated kinase phosphoserine/threonine/tyrosine binding protein) dramatically increases the number of what appeared to be primary neurites in rat pheochromocytoma (PC-12) cells; however, the question remained whether these MK-STYX-induced outgrowths were bona fide neurites, and formed synapses. Here, we report that microtubules and microfilaments, components of the cytoskeleton that are involved in the formation of neurites, are present in MK-STYX-induced outgrowths. In addition, in response to nerve growth factor (NGF), MK-STYX-expressing cells produced more growth cones than non-MK-STYX-expressing cells, further supporting a model in which MK-STYX has a role in actin signaling. Furthermore, immunoblot analysis demonstrates that MK-STYX modulates actin expression. Transmission electron microscopy confirmed that MK-STYX-induced neurites form synapses. To determine whether these MK-STYX-induced neurites have pre-synaptic or post-synaptic properties, we used classical markers for axons and dendrites, Tau-1 and MAP2 (microtubule associated protein 2), respectively. MK-STYX induced neurites were dopaminergic and expression of both Tau-1 and MAP2 suggests that they have both axonal and dendritic properties. Further studies in rat hippocampal primary neurons demonstrated that MK-STYX altered their morphology. A significant number of primary neurons in the presence of MK-STYX had more than the normal number of primary neurites. Our data illustrate the novel findings that MK-STYX induces outgrowths in PC-12 cells that fit the criteria for neurites, have a greater number of growth cones, form synapses, and have pre-synaptic and post-synaptic properties. It also highlights that the pseudophosphatase MK-STYX significantly alters the morphology of primary neurons.

Antagonistic roles for STYX pseudophosphatases in neurite outgrowth.

Mitogen-activated protein kinases (MAPKs) are essential players in important neuronal signaling pathways including neuronal development, plasticity, survival, learning, and memory. The inactivation of MAPKs is tightly controlled by MAPK phosphatases (MKPs), which also are important regulators of these neuronal processes. Considering that MAPKs and MKPs are major players in neuronal signaling, it follows that their misregulation is pivotal in neurodegenerative diseases such as Alzheimer's, Huntington's, Parkinson's, and amyotrophic lateral sclerosis. In contrast, the actions of their noncatalytic homologs, or pseudoenzymes, have received minimal attention as important regulators in neuronal signaling pathways and relevant diseases. There is compelling evidence, however, that pseudophosphatases, such as STYX (phospho-serine-threonine/tyrosine-binding protein) and MAPK-STYX (MK-STYX), are integral signaling molecules in regulating pathways involved in neuronal developmental processes such as neurite outgrowth. Here, we discuss how the dynamics of MK-STYX in the stress response pathway imply that this unique member of the MKP subfamily has the potential to have a major role in neuronal signaling. We further compare the actions of STYX in preventing neurite-like outgrowths and MK-STYX in inducing neurite outgrowths. The roles of these pseudophosphatases in neurite outgrowth highlight their emergence as important candidates to investigate in neurodegenerative disorders and diseases.

Analyzing Pseudophosphatase Function.

Pseudophosphatases regulate signal transduction cascades, but their mechanisms of action remain enigmatic. Reflecting this mystery, the prototypical pseudophosphatase STYX (phospho-serine-threonine/tyrosine-binding protein) was named with allusion to the river of the dead in Greek mythology to emphasize that these molecules are "dead" phosphatases. Although proteins with STYX domains do not catalyze dephosphorylation, this in no way precludes their having other functions as integral elements of signaling networks. Thus, understanding their roles in signaling pathways may mark them as potential novel drug targets. This chapter outlines common strategies used to characterize the functions of pseudophosphatases, using as an example MK-STYX [mitogen-activated protein kinase (MAPK) phospho-serine-threonine/tyrosine binding], which has been linked to tumorigenesis, apoptosis, and neuronal differentiation. We start with the importance of "restoring" (when possible) phosphatase activity in a pseudophosphatase so that the active mutant may be used as a comparison control throughout immunoprecipitation and mass spectrometry analyses. To this end, we provide protocols for site-directed mutagenesis, mammalian cell transfection, co-immunoprecipitation, phosphatase activity assays, and immunoblotting that we have used to investigate MK-STYX and the active mutant MK-STYXactive. We also highlight the importance of utilizing RNA interference (RNAi) "knockdown" technology to determine a cellular phenotype in various cell lines. Therefore, we outline our protocols for introducing short hairpin RNA (shRNA) expression plasmids into mammalians cells and quantifying knockdown of gene expression with real-time quantitative PCR (qPCR). A combination of cellular, molecular, biochemical, and proteomic techniques has served as powerful tools in identifying novel functions of the pseudophosphatase MK-STYX. Likewise, the information provided here should be a helpful guide to elucidating the function of other pseudophosphatases.

The pseudophosphatase MK-STYX induces neurite-like outgrowths in PC12 cells.

The rat pheochromocytoma PC12 cell line is a widely used system to study neuronal differentiation for which sustained activation of the extracellular signaling related kinase (ERK) pathway is required. Here, we investigate the function of MK-STYX [MAPK (mitogen-activated protein kinase) phosphoserine/threonine/tyrosine-binding protein] in neuronal differentiation. MK-STYX is a member of the MAPK phosphatase (MKP) family, which is generally responsible for dephosphorylating the ERKs. However, MK-STYX lacks catalytic activity due to the absence of the nucleophilic cysteine in the active site signature motif HC(X5)R that is essential for phosphatase activity. Despite being catalytically inactive, MK-STYX has been shown to play a role in important cellular pathways, including stress responses. Here we show that PC12 cells endogenously express MK-STYX. In addition, MK-STYX, but not its catalytically active mutant, induced neurite-like outgrowths in PC12 cells. Furthermore, MK-STYX dramatically increased the number of cells with neurite extensions in response to nerve growth factor (NGF), whereas the catalytically active mutant did not. MK-STYX continued to induce neurites in the presence of a MEK (MAP kinase kinase) inhibitor suggesting that MK-STYX does not act through the Ras-ERK/MAPK pathway but is involved in another pathway whose inactivation leads to neuronal differentiation. RhoA activity assays indicated that MK-STYX induced extensions through the Rho signaling pathway. MK-STYX decreased RhoA activation, whereas RhoA activation increased when MK-STYX was down-regulated. Furthermore, MK-STYX affected downstream players of RhoA such as the actin binding protein cofilin. The presence of MK-STYX decreased the phosphorylation of cofilin in non NGF stimulated cells, but increased its phosphorylation in NGF stimulated cells, whereas knocking down MK-STYX caused an opposite effect. Taken together our data suggest that MK-STYX may be a regulator of RhoA signaling, and implicate this pseudophosphatase as a regulator of neuronal differentiation.

The pseudophosphatase MK-STYX inhibits stress granule assembly independently of Ser149 phosphorylation of G3BP-1.

The pseudophosphatase MK-STYX (mitogen-activated protein kinase phosphoserine/threonine/tyrosine-binding protein) has been implicated in the stress response pathway. The expression of MK-STYX inhibits the assembly of stress granules, which are cytoplasmic storage sites for mRNA that form as a protective mechanism against stressors such as heat shock, UV irradiation and hypoxia. Furthermore, MK-STYX interacts with a key component of stress granules: G3BP-1 (Ras-GTPase activating protein SH3 domain binding protein-1). Because G3BP-1 dephosphorylation at Ser149 induces stress granule assembly, we initially hypothesized that the inhibition of stress granules by MK-STYX was G3BP-1 phosphorylation-dependent. However, in the present study, using MK-STYX constructs and G3BP-1 phosphomimetic or nonphosphorylatable mutants, we show that MK-STYX inhibits stress granule formation independently of G3BP-1 phosphorylation at Ser149. The introduction of point mutations at the 'active site' of MK-STYX that convert serine and phenylalanine to histidine and cysteine, respectively, is sufficient to generate an active enzyme. In separate experiments, we show that this active mutant, MK-STYX(active), has opposite effects to wild-type MK-STYK. Not only does MK-STYX(active) induce stress granules, but also it has the capacity to dephosphorylate G3BP-1. Taken together, these results provide evidence that the pseudophosphatase MK-STYX plays a key role in the cellular response to stress.

Recruitment of the oncoprotein v-ErbA to aggresomes.

Aggresome formation, a cellular response to misfolded protein aggregates, is linked to cancer and neurodegenerative disorders. Previously we showed that Gag-v-ErbA (v-ErbA), a retroviral variant of the thyroid hormone receptor (TRα1), accumulates in and sequesters TRα1 into cytoplasmic foci. Here, we show that foci represent v-ErbA targeting to aggresomes. v-ErbA colocalizes with aggresomal markers, proteasomes, hsp70, HDAC6, and mitochondria. Foci have hallmark characteristics of aggresomes: formation is microtubule-dependent, accelerated by proteasome inhibitors, and they disrupt intermediate filaments. Proteasome-mediated degradation is critical for clearance of v-ErbA and T(3)-dependent TRα1 clearance. Our studies highlight v-ErbA's complex mode of action: the oncoprotein is highly mobile and trafficks between the nucleus, cytoplasm, and aggresome, carrying out distinct activities within each compartment. Dynamic trafficking to aggresomes contributes to the dominant negative activity of v-ErbA and may be enhanced by the viral Gag sequence. These studies provide insight into novel modes of oncogenesis across multiple cellular compartments.

The pseudophosphatase MK-STYX interacts with G3BP and decreases stress granule formation.

MK-STYX [MAPK (mitogen-activated protein kinase) phospho-serine/threonine/tyrosine-binding protein] is a pseudophosphatase member of the dual-specificity phosphatase subfamily of the PTPs (protein tyrosine phosphatases). MK-STYX is catalytically inactive due to the absence of two amino acids from the signature motif that are essential for phosphatase activity. The nucleophilic cysteine residue and the adjacent histidine residue, which are conserved in all active dual-specificity phosphatases, are replaced by serine and phenylalanine residues respectively in MK-STYX. Mutations to introduce histidine and cysteine residues into the active site of MK-STYX generated an active phosphatase. Using MS, we identified G3BP1 [Ras-GAP (GTPase-activating protein) SH3 (Src homology 3) domain-binding protein-1], a regulator of Ras signalling, as a binding partner of MK-STYX. We observed that G3BP1 bound to native MK-STYX; however, binding to the mutant catalytically active form of MK-STYX was dramatically reduced. G3BP1 is also an RNA-binding protein with endoribonuclease activity that is recruited to 'stress granules' after stress stimuli. Stress granules are large subcellular structures that serve as sites of mRNA sorting, in which untranslated mRNAs accumulate. We have shown that expression of MK-STYX inhibited stress granule formation induced either by aresenite or expression of G3BP itself; however, the catalytically active mutant MK-STYX was impaired in its ability to inhibit G3BP-induced stress granule assembly. These results reveal a novel facet of the function of a member of the PTP family, illustrating a role for MK-STYX in regulating the ability of G3BP1 to integrate changes in growth-factor stimulation and environmental stress with the regulation of protein synthesis.

Regulation of cleavage by protein kinase C in Chaetopterus.

We report that protein kinase C (PKC) plays a regulatory role in early cleavage in Chaetopterus eggs. Using Western blotting, we assayed the expression patterns of conventional PKCs (cPKC), novel PKCs (nPKC), and atypical PKCs (aPKC). During early development after fertilization, PKC protein levels varied independently by isoform. PKC protein expression during differentiation, without cleavage and after parthenogenetic activation, was very similar to that during normal development indicating that PKC gene expression does not require cellularization. Since PKC has been shown to regulate meiosis in this organism, we also assayed the membrane association of these isoforms as an indicator of their activation during meiosis and early cleavage. PKC-gamma transiently associated with membranes and therefore became activated before meiotic division and cleavage, whereas PKC-alpha and -beta transiently dissociated from membranes and therefore became inactivated at these times. Inhibition of these PKC isoforms by bisindolylmaleimide I had no effect on cleavage or early development to the trochophore larva, indicating that PKC-gamma activation is not essential for cleavage or early development. However, their persistent activation by thymeleatoxin blocked cleavage. The results indicate that the dissociation of PKC-alpha and/or -beta from the membrane fraction, and therefore their inactivation, is essential for normal cleavage. Elevated PKC activity is essential for nuclear envelope breakdown and spindle formation at meiosis I. By contrast, down-regulation of this activity is essential for cleavage after fertilization.

Protein tyrosine phosphatases in Chaetopterus egg activation.

Changes in protein tyrosine phosphorylation are an essential aspect of egg activation after fertilization. Such changes result from the net contributions of both tyrosine kinases and phosphatases (PTP). This study was conducted to determine what role(s) PTP may have in egg activation. We identified four novel PTP in Chaetopterus pergamentaceus oocytes, cpPTPNT6, cpPTPNT7, cpPTPR2B, and cpPTPR2A, that have significant homology to, respectively, human PTPsigma, -rho, -D2 and -BAS. The first two are cytosolic and the latter two are transmembrane. Several PTP inhibitors were tested to see if they would affect Chaetopterus pergamentaceus fertilization. Eggs treated with beta-bromo-4-hydroxyacetophenone (PTP inhibitor 1) exhibited microvillar elongation, which is a sign of cortical changes resulting from activation. Those treated with Na3VO4 underwent full parthenogenetic activation, including polar body formation and pseudocleavage and did so independently of extracellular Ca2+, which is required for the Ca2+ oscillations that initiate development after fertilization. Fluorescence microscopy identified phosphotyrosine-containing proteins in the cortex and around the nucleus of vanadate-activated eggs, whereas in fertilized eggs they were concentrated only in the cortex. Immunoblots of vanadate-activated and fertilized eggs showed tyrosine hyperphosphorylation of approximately 140 kDa protein. These results suggest that PTP most likely maintain the egg in an inactive state by dephosphorylation of proteins independent of the Ca2+ oscillations in the activation process.