Glycogen synthase kinase-3 enhances nuclear export of a Dictyostelium STAT protein

Extracellular cAMP stimulates the rapid tyrosine phosphorylation and nuclear translocation of the DICTYOSTELIUM: STAT protein Dd-STATa. Here we show that it also induces serine phosphorylation by GskA, a homologue of glycogen synthase kinase-3 (GSK-3). Tyrosine phosphorylation occurs within 10 s of stimulation, whereas serine phosphorylation takes 5 min, matching the kinetics observed for the cAMP regulation of GskA. Phosphorylation by GskA enhances nuclear export of Dd-STATa. The phosphorylated region, however, is not itself a nuclear export signal and we identify a region elsewhere in the protein that mediates nuclear export. These results suggest a biphasic regulation of Dd-STATa, in which extracellular cAMP initially directs nuclear import and then, via GskA, promotes its subsequent export. It also raises the possibility of an analogous regulation of STAT nuclear export in higher eukaryotes.     

Protein tyrosine phosphatase PTP1 negatively regulates Dictyostelium STATa and is required for proper cell-type proportioning

The protein tyrosine phosphatase PTP1, which mediates reversible phosphorylation on tyrosine, has been shown to play an important regulatory role during Dictyostelium development. Mutants lacking PTP1 develop more rapidly than normal, while strains that overexpress PTP1 display aberrant morphology. However, the signalling pathways involved have not been characterised. In reexamining these strains, we have found that there is an inverse correlation between levels of PTP1 activity, the extent of tyrosine phosphorylation on Dictyostelium STATa after treatment with cAMP, and the proportion of the slug population exhibiting STATa nuclear enrichment in vivo. This suggests that PTP1 acts to attenuate the tyrosine phosphorylation of STATa and downstream STATa-mediated pathways. Consistent with this, we show that when PTP1 is overexpressed, there is increased expression of a prestalk cell marker at the slug posterior, a phenocopy of STATa null slugs. In ptp1 null strains, STATa tyrosine phosphorylation and nuclear enrichment in the slug anterior is increased. There is also a change in the prestalk to prespore cell ratio. Synergy experiments suggest that this is due to a cell-autonomous defect in forming the subset of prespore cells that are located in the anterior prespore region.     

Tyrosine phosphorylation of sam68 by breast tumor kinase regulates intranuclear localization and cell cycle progression

The breast tumor kinase (BRK) is a growth promoting non-receptor tyrosine kinase overexpressed in the majority of human breast tumors. BRK is known to potentiate the epidermal growth factor (EGF) response in these cells. Although BRK is known to phosphorylate the RNA-binding protein Sam68, the specific tyrosines phosphorylated and the exact role of this phosphorylation remains unknown. Herein, we have generated Sam68 phospho-specific antibodies against C-terminal phosphorylated tyrosine residues within the Sam68 nuclear localization signal. We show that BRK phosphorylates Sam68 on all three tyrosines in the nuclear localization signal. By indirect immunofluorescence we observed that BRK and EGF treatment not only phosphorylates Sam68 but also induces its relocalization. Tyrosine 440 was identified as a principal modulator of Sam68 localization and this site was phosphorylated in response to EGF treatment in human breast tumor cell lines. Moreover, this phosphorylation event was inhibited by BRK small interfering RNA treatment, consistent with Sam68 being a physiological substrate of BRK downstream of the EGF receptor in breast cancer cells. Finally, we observed that Sam68 suppressed BRK-induced cell proliferation, suggesting that Sam68 does indeed contain anti-proliferative properties that may be neutralized in breast cancer cells by phosphorylation.     

SNT, a differentiation-specific target of neurotrophic factor-induced tyrosine kinase activity in neurons and PC12 cells.

To elucidate the signal transduction mechanisms used by ligands that induce differentiation and the cessation of cell division, we utilized p13suc1-agarose, a reagent that binds p34cdc2/cdk2. By using this reagent, we identified a 78- to 90-kDa species in PC12 pheochromocytoma cells that is rapidly phosphorylated on tyrosine following treatment with the differentiation factors nerve growth factor (NGF) and fibroblast growth factor but not by the mitogens epidermal growth factor or insulin. This species, called SNT (suc-associated neurotrophic factor-induced tyrosine-phosphorylated target), was also phosphorylated on tyrosine in primary rat cortical neurons treated with the neurotrophic factors neurotrophin-3, brain-derived neurotrophic factor, and fibroblast growth factor but not in those treated with epidermal growth factor. In neuronal and fibroblast cells, where NGF can also act as a mitogen, SNT was tyrosine phosphorylated to a much greater extent during NGF-induced differentiation than during NGF-induced proliferation. SNT was phosphorylated in vitro on serine, threonine, and tyrosine in p13suc1-agarose precipitates from NGF-treated PC12 cells, indicating that this protein may be a substrate of kinase activities associated with p13suc1-p34cdc2/cdk2 complexes. In addition, SNT was associated predominantly with nuclear fractions following subcellular fractionation of NGF-treated PC12 cells. Finally, in PC12 cells, NGF-stimulated tyrosine phosphorylation of SNT was dependent on the levels of Trk tyrosine kinase activity and was constitutively induced by expression of pp60v-src. However, Ras was not required for constitutive SNT tyrosine phosphorylation, suggesting that this protein functions distally to Trk and pp60v-src but in a pathway parallel to that of Ras. SNT is the first identified specific target of differentiation factor-induced tyrosine kinase activity in neuronal cells.     

The aryl hydrocarbon receptor (AhR) tyrosine 9, a residue that is essential for AhR DNA binding activity, is not a phosphoresidue but augments AhR phosphorylation

We delineate a mechanism by which dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin or TCDD)-mediated formation of the aryl hydrocarbon receptor (AhR) DNA binding complex is disrupted by a single mutation at the conserved AhR tyrosine 9. Replacement of tyrosine 9 with the structurally conservative phenylalanine (AhRY9F) abolished binding to dioxin response element (DRE) D, E, and A and abrogated DRE-driven gene induction mediated by the AhR with no effect on TCDD binding, TCDD-induced nuclear localization, or ARNT heterodimerization. The speculated role for phosphorylation at tyrosine 9 was also examined. Anti-phosphotyrosine immunoblotting could not detect a major difference between the AhRY9F mutant and wild-type AhR, but a basic isoelectric point shift was detected by two-dimensional gel electrophoresis of AhRY9F. However, an antibody raised to recognize only phosphorylated tyrosine 9 (anti-AhRpY9) confirmed that AhR tyrosine 9 is not a phosphorylated residue required for DRE binding. Kinase assays using synthetic peptides corresponding to the wild-type and mutant AhR residues 1-23 demonstrated that a tyrosine at position 9 is important for substrate recognition at serine(s)/threonine(s) within this sequence by purified protein kinase C (PKC). Also, compared with AhRY9F, immunopurified full-length wild-type receptor was more rapidly phosphorylated by PKC. Furthermore, co-treatment of AhR-deficient cells that expressed AhRY9F and a DRE-driven luciferase construct with phorbol 12-myristate 13-acetate and TCDD resulted in a 30% increase in luciferase activity compared with AhRY9F treated with TCDD alone. Overall, AhR tyrosine 9, which is not a phosphorylated residue itself but is required for DNA binding, appears to play a crucial role in AhR activity by permitting proper phosphorylation of the AhR.     

p250GAP,a neural RhoGAP protein,is associated with and phosphorylated by Fyn

Fyn is a member of the Src-family protein tyrosine kinases and plays important roles in both neurons and oligodendrocytes. Here we report association of Fyn with p250GAP, a RhoGAP protein that is expressed predominantly in brain. p250GAP interacts with Fyn both in vitro and in vivo. p250GAP is tyrosine phosphorylated by Fyn when co-expressed in HEK293T cells. This phosphorylation appears to enhance the interaction between p250GAP and Fyn. Furthermore, the level of tyrosine phosphorylation of p250GAP increases upon differentiation of the oligodendrocyte cell line CG4. Given that Fyn activity is up-regulated during oligodendrocyte maturation, the results argue that p250GAP is phosphorylated by Fyn in oligodendrocytes. Tyrosine phosphorylation of p250GAP by Fyn would regulate its RhoGAP activity, subcellular localization, or interactions with other proteins, leading to morphological and phenotypic changes of oligodendrocytes.     

Cytoskeleton-dependent tyrosine phosphorylation of the p130(Cas) family member HEF1 downstream of the G protein-coupled calcitonin receptor. Calcitonin induces the association of HEF1, paxillin, and focal adhesion kinase.

HEF1 is a recently described p130(Cas)-like docking protein that contains one SH3 domain and multiple SH2 binding motifs. In B cells, HEF1 is phosphorylated by a cytoskeleton-dependent mechanism that is triggered by integrin ligation. However, the induction of HEF1 phosphorylation by G protein-coupled receptors has not been reported. We found that HEF1, but not p130(Cas), is tyrosine-phosphorylated following stimulation of the rabbit C1a calcitonin receptor stably expressed in HEK-293 cells. The calcitonin-induced tyrosine phosphorylation of HEF1 increased in a time- and dose-dependent manner. Dibutyryl cAMP and forskolin had little or no effect on HEF1 phosphorylation, and the protein kinase A inhibitor H89 failed to detectably inhibit the response to calcitonin, indicating that the G(s)/cAMP/protein kinase A pathway does not mediate the calcitonin effect. Pertussis toxin, which selectively blocks G(i/o) signaling, also had no effect. Increasing cytosolic Ca(2+) with ionomycin stimulated HEF1 phosphorylation and preventing any calcitonin-induced change in cytosolic calcium by a combination of BAPTA and extracellular EGTA completely blocked the calcitonin-induced tyrosine phosphorylation of HEF1. Phorbol 12-myristate 13-acetate also induced HEF1 tyrosine phosphorylation, and the protein kinase C inhibitor calphostin C completely inhibited both calcitonin- and phorbol 12-myristate 13-acetate-stimulated HEF1 phosphorylation. Calcitonin also induced the tyrosine phosphorylation of paxillin and focal adhesion kinase, and the association of these two proteins with HEF1. Pretreatment with cytochalasin D, which disrupts actin microfilaments, prevented the calcitonin-induced HEF1 and paxillin phosphorylation. In conclusion, the calcitonin-stimulated tyrosine phosphorylation of HEF1 is mediated by calcium- and protein kinase C-dependent mechanisms and requires the integrity of the actin cytoskeleton.     

Focal adhesion kinase regulation of N-WASP subcellular localization and function

N-WASP is a member of the WASP family of proteins that regulate actin cytoskeleton remodeling. FAK is a cytoplasmic tyrosine kinase implicated in integrin signaling during cell migration. Here we identify a direct interaction between N-WASP and FAK and show that N-WASP is phosphorylated by FAK at a conserved tyrosine residue, Tyr(256). We found that phosphorylation of Tyr(256) affected N-WASP nuclear localization, suggesting that phosphorylation of N-WASP by FAK may regulate its activity in vivo by altering its subcellular localization. We also showed that the nuclear localization of N-WASP is dependent on its being in the open conformation either after its activation by Cdc42 or the truncation of the C-terminal VCA domain. Phosphorylation of Tyr(256) of N-WASP could reduce its interaction with nuclear importin NPI-1, which might be responsible for its decreased nuclear localization. Lastly, we show that phosphorylation of Tyr(256) plays an important role in promoting cell migration. Together, these results suggest a novel regulatory mechanism of N-WASP by tyrosine phosphorylation and subcellular localization and its potential role in the regulation of cell migration.     

CDK-Dependent Nuclear Localization of B-Cyclin Clb1 Promotes FEAR Activation during Meiosis I in Budding Yeast

Cyclin-dependent kinases (CDK) are master regulators of the cell cycle in eukaryotes. CDK activity is regulated by the presence, post-translational modification and spatial localization of its regulatory subunit cyclin. In budding yeast, the B-cyclin Clb1 is phosphorylated and localizes to the nucleus during meiosis I. However the functional significance of Clb1''s phosphorylation and nuclear localization and their mutual dependency is unknown. In this paper, we demonstrate that meiosis-specific phosphorylation of Clb1 requires its import to the nucleus but not vice versa. While Clb1 phosphorylation is dependent on activity of both CDK and polo-like kinase Cdc5, its nuclear localization requires CDK but not Cdc5 activity. Furthermore we show that increased nuclear localization of Clb1 during meiosis enhances activation of FEAR (Cdc Fourteen Early Anaphase Release) pathway. We discuss the significance of our results in relation to regulation of exit from meiosis I.     

Distinct protein tyrosine phosphorylation during mitogenesis induced in quiescent sv40-transformed 3T3 T cells by insulin or vanadate

Insulin and vanadate selectively induce mitogenesis in quiescent SV40 large T antigen-transformed 3T3 T cells (CSV3–1) but not in quiescent nontransformed 3T3 T cells. Insulin and vanadate mediate this effect in CSV3–1 cells by distinct signal transduction mechanisms that involve protein tyrosine kinase activity. To further study these processes, changes in protein tyrosine phosphorylation induced by insulin and vanadate were investigated. Using immunoprecipitation and Western blotting techniques with antiphosphotyrosine antibodies, we report distinct protein phosphorylation characteristics in insulin- and vanadate-stimulated CSV3–1 cells. The insulin receptor β-subunit is phosphorylated within 2 min after insulin stimulation of transformed CSV3–1 cells. Insulin also stimulates a rapid increase in tyrosine phosphorylation of the 170 kDa insulin receptor substrate-1 and complex formation between the phosphorylated insulin receptor substrate-1 and the 85 kDa subunit of phosphatidylinositol 3'-kinase. In contrast, vanadate does not initially increase detectable phosphorylation of any proteins, including neither the insulin receptor nor the insulin receptor substrate-1. After 60 min, however, a marked increase in tyrosine phosphorylation of 55 and 64 kDa proteins is observed in vanadate-treated CSV3–1 cells. Furthermore, treatment of CSV3–1 cells with genistein abolishes the effects of vanadate on protein tyrosine phosphorylation but only minimally inhibits the effects of insulin. Finally, insulin stimulates the phosphorytion of a 33 kDa protein, whereas vanadate does not. By comparison, in nontransformed 3T3 T cells, insulin induces a delayed and weaker tyrosine phosphorylation of the insulin receptor β-subunit and vanadate does not enhance the tyrosine phosphorylation of the 55 and 64 kDa proteins. These data together indicate that the mitogenic effects of insulin and vanadate are associated with distinct protein phosphorylation patterns that appear to be differentially regulated in SV40-transformed and nontransformed 3T3 T cells. © 1994 Wiley-Liss, Inc.     

Dephosphorylation at a Conserved SP Motif Governs cAMP Sensitivity and Nuclear Localization of Class IIa Histone Deacetylases

Histone deacetylase 4 (HDAC4) and its paralogs, HDAC5, -7, and -9 (all members of class IIa), possess multiple phosphorylation sites crucial for 14-3-3 binding and subsequent nuclear export. cAMP signaling stimulates nuclear import of HDAC4 and HDAC5, but the underlying mechanisms remain to be elucidated. Here we show that cAMP potentiates nuclear localization of HDAC9. Mutation of an SP motif conserved in HDAC4, -5, and -9 prevents cAMP-stimulated nuclear localization. Unexpectedly, this treatment inhibits phosphorylation at the SP motif, indicating an inverse relationship between the phosphorylation event and nuclear import. Consistent with this, leptomycin B-induced nuclear import and adrenocorticotropic hormone (ACTH) treatment result in the dephosphorylation at the motif. Moreover, the modification synergizes with phosphorylation at a nearby site, and similar kinetics was observed for both phosphorylation events during myoblast and adipocyte differentiation. These results thus unravel a previously unrecognized mechanism whereby cAMP promotes dephosphorylation and differentially regulates multisite phosphorylation and the nuclear localization of class IIa HDACs.     

Tyrosine-phosphorylated extracellular signal--regulated kinase associates with the Golgi complex during G2/M phase of the cell cycle: evidence for regulation of Golgi structure.

Phosphorylation of the extracellular signal-regulated kinases (ERKs) on tyrosine and threonine residues within the TEY tripeptide motif induces ERK activation and targeting of substrates. Although it is recognized that phosphorylation of both residues is required for ERK activation, it is not known if a single phosphorylation of either residue regulates physiological functions. In light of recent evidence indicating that ERK proteins regulate substrate function in the absence of ERK enzymatic activity, we have begun to examine functional roles for partially phosphorylated forms of ERK. Using phosphorylation site--specific ERK antibodies and immunofluorescence, we demonstrate that ERK phosphorylated on the tyrosine residue (pY ERK) within the TEY activation sequence is found constitutively in the nucleus, and localizes to the Golgi complex of cells that are in late G2 or early mitosis of the cell cycle. As cells progress through metaphase and anaphase, pY ERK localization to Golgi vesicles is most evident around the mitotic spindle poles. During telophase, pY ERK associates with newly formed Golgi vesicles but is not found on there after cytokinesis and entry into G1. Increased ERK phosphorylation causes punctate distribution of several Golgi proteins, indicating disruption of the Golgi structure. This observation is reversible by overexpression of a tyrosine phosphorylation--defective ERK mutant, but not by a kinase-inactive ERK2 mutant that is tyrosine phosphorylated. These data provide the first evidence that pY ERK and not ERK kinase activity regulates Golgi structure and may be involved in mitotic Golgi fragmentation and reformation.     

Hyperosmotic stress induces rapid focal adhesion kinase phosphorylation at tyrosines 397 and 577. Role of Src family kinases and Rho family GTPases

Hyperosmotic stress induced by treatment of Swiss 3T3 cells with the non-permeant solutes sucrose or sorbitol, rapidly and robustly stimulated endogenous focal adhesion kinase (FAK) phosphorylation at Tyr-397, the major autophosphorylation site, and at Tyr-577, within the kinase activation loop. Hyperosmotic stress-stimulated FAK phosphorylation at Tyr-397 occurred via a Src-independent pathway, whereas Tyr-577 phosphorylation was completely blocked by exposure to the Src family kinase inhibitor PP-2. Inhibition of p38 MAP kinase or phosphatidylinositol 3-kinases did not prevent FAK phosphorylation stimulated by hyperosmotic stress. Overexpression of N17 RhoA did not reduce hyperosmotic stress-mediated localization of phosphorylated FAK to focal contacts and treatment with the Rho-associated kinase inhibitor Y-27632 did not prevent FAK translocation and tyrosine phosphorylation in response to hyperosmotic stress. Overexpression of N17 Rac only slightly altered the hyperosmotic stress-mediated localization of phosphorylated FAK to focal contacts. In contrast, overexpression of the N17 mutant of Cdc42 disrupted hyperosmotic stress-stimulated FAK Tyr-397 localization to focal contacts. Additionally, treatment of cells with Clostridium difficile toxin B potently inhibited hyperosmotic stress-induced FAK tyrosine phosphorylation. Furthermore, FAK null fibroblasts compared with their FAK containing controls show markedly increased sensitivity, manifest by subsequent apoptosis, to sustained hyperosmotic stress. Our results indicate that FAK plays a fundamental role in protecting cells from hyperosmotic stress, and that the pathway(s) that mediates FAK autophosphorylation at Tyr-397 in response to osmotic stress can be distinguished from the pathways utilized by many other stimuli, including neuropeptides and bioactive lipids (Rho- and Rho-associated kinase-dependent), tyrosine kinase receptor agonists (phosphatidylinositol 3-kinase-dependent), and integrins (Src-dependent).     

Phototaxis during the slug stage of Dictyostelium discoideum: a model study

During the slug stage, the cellular slime mould Dictyostelium discoideum moves towards light sources. We have modelled this phototactic behaviour using a hybrid cellular automata/partial differential equation model. In our model, individual amoebae are not able to measure the direction from which the light comes, and differences in light intensity do not lead to differentiation in motion velocity among the amoebae. Nevertheless, the whole slug orientates itself towards the light. This behaviour is mediated by a modification of the cyclic AMP (cAMP) waves. As an explanation for phototaxis, we propose the following mechanism, which is basically characterized by four processes: (i) light is focused on the distal side of the slug as a result of the so-called ''lens-effect''; (ii) differences in luminous intensity cause differences in NH₃ concentration; (iii) NH₃ alters the excitablity of the cell, and thereby the shape of the cAMP wave; and (iv) chemotaxis towards cAMP causes the slug to turn. We show that this mechanism can account for a number of other behaviours that have been observed in experiments, such as bidirectional phototaxis and the cancellation of bidirectionality by a decrease in the light intensity or the addition of charcoal to the medium.