Transforming growth factor β targeted inactivation of cyclin E:cyclin-dependent kinase 2 (Cdk2) complexes by inhibition of Cdk2 activating kinase activity

Transforming growth factor β (TGF-β)-mediated G1 arrest previously has been shown to specifically target inactivation of cyclin D:cyclin-dependent kinase (Cdk) 4/6 complexes. We report here that TGF-β-treated human HepG2 hepatocellular carcinoma cells arrest in G1, but retain continued cyclin D:Cdk4/6 activity and active, hypophosphorylated retinoblastoma tumor suppressor protein. Consistent with this observation, TGF-β-treated cells failed to induce p15INK4b, down-regulate CDC25A, or increase levels of p21CIP1, p27KIP1, and p57KIP2. However, TGF-β treatment resulted in the specific inactivation of cyclin E:Cdk2 complexes caused by absence of the activating Thr160 phosphorylation on Cdk2. Whole-cell lysates from TGF-β-treated cells showed inhibition of Cdk2 Thr160 Cdk activating kinase (CAK) activity; however, cyclin H:Cdk7 activity, a previously assumed mammalian CAK, was not altered. Saccharomyces cerevisiae contains a genetically and biochemically proven CAK gene, CAK1, that encodes a monomeric 44-kDa Cak1p protein unrelated to Cdk7. Anti-Cak1p antibodies cross-reacted with a 45-kDa human protein with CAK activity that was specifically down-regulated in response to TGF-β treatment. Taken together, these observations demonstrate that TGF-β signaling mediates a G1 arrest in HepG2 cells by targeting Cdk2 CAK and suggests the presence of at least two mammalian CAKs: one specific for Cdk2 and one for Cdk4/6.

T helper type 2 inflammatory disease in the absence of interleukin 4 and transcription factor STAT6

An important signaling pathway for the differentiation of T helper type 2 (TH2) cells from uncommitted CD4 T cell precursors is activation of the STAT6 transcription factor by interleukin 4 (IL-4). The protooncogene BCL-6 is also involved in TH2 differentiation, as BCL-6 −/− mice develop an inflammation of the heart and lungs associated with an overproduction of TH2 cells. Surprisingly, IL-4 −/− BCL-6 −/− and STAT6 −/− BCL-6 −/− double-mutant mice developed the same TH2-type inflammation of the heart and lungs as is characteristic of BCL-6 −/− mice. Furthermore, a TH2 cytokine response developed in STAT6 −/− BCL-6 −/− and IL-4 −/− BCL-6 −/− mice after immunization with a conventional antigen in adjuvant. In contrast to these in vivo findings, STAT6 was required for the in vitro differentiation of BCL-6 −/− T cells into TH2 cells. BCL-6, a transcriptional repressor that can bind to the same DNA binding motifs as STAT transcription factors, seems to regulate TH2 responses in vivo by a pathway independent of IL-4 and STAT6.

Cluster of Differentiation Antigen 4 (CD4) Endocytosis and Adaptor Complex Binding Require Activation of the CD4 Endocytosis Signal by Serine Phosphorylation

Cluster of differentiation antigen 4 (CD4), the T lymphocyte antigen receptor component and human immunodeficiency virus coreceptor, is down-modulated when cells are activated by antigen or phorbol esters. During down-modulation CD4 dissociates from p56lck, undergoes endocytosis through clathrin-coated pits, and is then sorted in early endosomes to late endocytic organelles where it is degraded. Previous studies have suggested that phosphorylation and a dileucine sequence are required for down-modulation. Using transfected HeLa cells, in which CD4 endocytosis can be studied in the absence of p56lck, we show that the dileucine sequence in the cytoplasmic domain is essential for clathrin-mediated CD4 endocytosis. However, this sequence is only functional as an endocytosis signal when neighboring serine residues are phosphorylated. Phosphoserine is required for rapid endocytosis because CD4 molecules in which the cytoplasmic domain serine residues are substituted with glutamic acid residues are not internalized efficiently. Using surface plasmon resonance, we show that CD4 peptides containing the dileucine sequence bind weakly to clathrin adaptor protein complexes 2 and 1. The affinity of this interaction is increased 350- to 700-fold when the peptides also contain phosphoserine residues.

Thrombospondin-1 Induces Tyrosine Phosphorylation of Adherens Junction Proteins and Regulates an Endothelial Paracellular Pathway

Thrombospondin-1 (TSP) induces endothelial cell (EC) actin reorganization and focal adhesion disassembly and influences multiple EC functions. To determine whether TSP might regulate EC–EC interactions, we studied the effect of exogenous TSP on the movement of albumin across postconfluent EC monolayers. TSP increased transendothelial albumin flux in a dose-dependent manner at concentrations ≥1 μg/ml (2.2 nM). Increases in albumin flux were observed as early as 1 h after exposure to 30 μg/ml (71 nM) TSP. Inhibition of tyrosine kinases with herbimycin A or genistein protected against the TSP-induced barrier dysfunction by >80% and >50%, respectively. TSP-exposed monolayers exhibited actin reorganization and intercellular gap formation, whereas pretreatment with herbimycin A protected against this effect. Increased staining of phosphotyrosine-containing proteins was observed in plaque-like structures and at the intercellular boundaries of TSP-treated cells. In the presence of protein tyrosine phosphatase inhibition, TSP induced dose- and time-dependent increments in levels of phosphotyrosine-containing proteins; these TSP dose and time requirements were compatible with those defined for EC barrier dysfunction. Phosphoproteins that were identified include the adherens junction proteins focal adhesion kinase, paxillin, γ-catenin, and p120Cas. These combined data indicate that TSP can modulate endothelial barrier function, in part, through tyrosine phosphorylation of EC proteins.

Insulin-induced Stimulation of Na+,K+-ATPase Activity in Kidney Proximal Tubule Cells Depends on Phosphorylation of the α-Subunit at Tyr-10

Phosphorylation of the α-subunit of Na+,K+-ATPase plays an important role in the regulation of this pump. Recent studies suggest that insulin, known to increase solute and fluid reabsorption in mammalian proximal convoluted tubule (PCT), is stimulating Na+,K+-ATPase activity through the tyrosine phosphorylation process. This study was therefore undertaken to evaluate the role of tyrosine phosphorylation of the Na+,K+-ATPase α-subunit in the action of insulin. In rat PCT, insulin and orthovanadate (a tyrosine phosphatase inhibitor) increased tyrosine phosphorylation level of the α-subunit more than twofold. Their effects were not additive, suggesting a common mechanism of action. Insulin-induced tyrosine phosphorylation was prevented by genistein, a tyrosine kinase inhibitor. The site of tyrosine phosphorylation was identified on Tyr-10 by controlled trypsinolysis in rat PCTs and by site-directed mutagenesis in opossum kidney cells transfected with rat α-subunit. The functional relevance of Tyr-10 phosphorylation was assessed by 1) the abolition of insulin-induced stimulation of the ouabain-sensitive 86Rb uptake in opossum kidney cells expressing mutant rat α1-subunits wherein tyrosine was replaced by alanine or glutamine; and 2) the similarity of the time course and dose dependency of the insulin-induced increase in ouabain-sensitive 86Rb uptake and tyrosine phosphorylation. These findings indicate that phosphorylation of the Na+,K+-ATPase α-subunit at Tyr-10 likely participates in the physiological control of sodium reabsorption in PCT.

Activation and Functional Analysis of Janus Kinase 2 in BA/F3 Cells Using the Coumermycin/Gyrase B System

Janus kinase 2 (Jak2) protein tyrosine kinase plays an important role in interleukin-3– or granulocyte–macrophage colony-stimulating factor–mediated signal transduction pathways leading to cell proliferation, activation of early response genes, and inhibition of apoptosis. However, it is unclear whether Jak2 can activate these signaling pathways directly without the involvement of cytokine receptor phosphorylation. To investigate the specific role of Jak2 in the regulation of signal transduction pathways, we generated gyrase B (GyrB)–Jak2 fusion proteins, dimerized through the addition of coumermycin. Coumermycin induced autophosphorylation of GyrB–Jak2 fusion proteins, thus bypassing receptor activation. Using different types of chimeric Jak2 molecules, we observed that although the kinase domain of Jak2 is sufficient for autophosphorylation, the N-terminal regions are essential for the phosphorylation of Stat5 and for the induction of short-term cell proliferation. Moreover, coumermycin-induced activation of Jak2 can also lead to increased levels of c-myc and CIS mRNAs in BA/F3 cells stably expressing the Jak2 fusion protein with the intact N-terminal region. Conversely, activation of the chimeric Jak2 induced neither phosphorylation of Shc or SHP-2 nor activation of the c-fos promoter. Here, we showed that the GyrB–Jak2 system can serve as an excellent model to dissect signals of receptor-dependent and -independent events. We also obtained evidence indicating a role for the N-terminal region of Jak2 in downstream signaling events.

Extracellular Calcium Regulates HeLa Cell Morphology during Adhesion to Gelatin: Role of Translocation and Phosphorylation of Cytosolic Phospholipase A2

Attachment of HeLa cells to gelatin induces the release of arachidonic acid (AA), which is essential for cell spreading. HeLa cells spreading in the presence of extracellular Ca2+ released more AA and formed more distinctive lamellipodia and filopodia than cells spreading in the absence of Ca2+. Addition of exogenous AA to cells spreading in the absence of extracellular Ca2+ restored the formation of lamellipodia and filopodia. To investigate the role of cytosolic phospholipase A2 (cPLA2) in regulating the differential release of AA and subsequent formation of lamellipodia and filopodia during HeLa cell adhesion, cPLA2 phosphorylation and translocation from the cytosol to the membrane were evaluated. During HeLa cell attachment and spreading in the presence of Ca2+, all cPLA2 became phosphorylated within 2 min, which is the earliest time cell attachment could be measured. In the absence of extracellular Ca2+, the time for complete cPLA2 phosphorylation was lengthened to <4 min. Maximal translocation of cPLA2 from cytosol to membrane during adhesion of cells to gelatin was similar in the presence or absence of extracellular Ca2+ and remained membrane associated throughout the duration of cell spreading. The amount of total cellular cPLA2 translocated to the membrane in the presence of extracellular Ca2+ went from <20% for unspread cells to >95% for spread cells. In the absence of Ca2+ only 55–65% of the total cPLA2 was translocated to the membrane during cell spreading. The decrease in the amount translocated could account for the comparable decrease in the amount of AA released by cells during spreading without extracellular Ca2+. Although translocation of cPLA2 from cytosol to membrane was Ca2+ dependent, phosphorylation of cPLA2 was attachment dependent and could occur both on the membrane and in the cytosol. To elucidate potential activators of cPLA2, the extracellular signal-related protein kinase 2 (ERK2) and protein kinase C (PKC) were investigated. ERK2 underwent a rapid phosphorylation upon early attachment followed by a dephosphorylation. Both rates were enhanced during cell spreading in the presence of extracellular Ca2+. Treatment of cells with the ERK kinase inhibitor PD98059 completely inhibited the attachment-dependent ERK2 phosphorylation but did not inhibit cell spreading, cPLA2 phosphorylation, translocation, or AA release. Activation of PKC by phorbol ester (12-O-tetradecanoylphorbol-13-acetate) induced and attachment-dependent phosphorylation of both cPLA2 and ERK2 in suspension cells. However, in cells treated with the PKC inhibitor Calphostin C before attachment, ERK2 phosphorylation was inhibited, whereas cPLA2 translocation and phosphorylation remained unaffected. In conclusion, although cPLA2-mediated release of AA during HeLa cell attachment to a gelatin substrate was essential for cell spreading, neither ERK2 nor PKC appeared to be responsible for the attachment-induced cPLA2 phosphorylation and the release of AA.

Two Distinct Signaling Pathways Downstream of Janus Kinase 2 Play Redundant Roles for Antiapoptotic Activity of Granulocyte-Macrophage Colony-stimulating Factor

Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) induces proliferation and sustains the viability of the mouse interleukin-3-dependent cell line BA/F3 expressing the hGM-CSF receptor. Analysis of the antiapoptosis activity of GM-CSF receptor βc mutants showed that box1 but not the C-terminal region containing tyrosine residues is essential for GM-CSF-dependent antiapoptotic activity. Because βc mutants, which activate Janus kinase 2 but neither signal transducer and activator of transcription 5 nor the MAPK cascade sustain antiapoptosis activity, involvement of Janus kinase 2, excluding the above molecules, in antiapoptosis activity seems likely. GM-CSF activates phosphoinositide-3-OH kinase as well as Akt, and activation of both was suppressed by addition of wortmannin. Interestingly, wortmannin did not affect GM-CSF-dependent antiapoptosis, thus indicating that the phosphoinositide-3-OH kinase pathway is not essential for cell surivival. Analysis using the tyrosine kinase inhibitor genistein and a MAPK/extracellular signal-regulated kinase (ERK) kinase 1 inhibitor, PD98059, indicates that activation of either the genistein-sensitive signaling pathway or the PD98059-sensitive signaling pathway from βc may be sufficient to suppress apoptosis. Wild-type and a βc mutant lacking tyrosine residues can induce expression of c-myc and bcl-xL genes; however, drug sensitivities for activation of these genes differ from those for antiapoptosis activity of GM-CSF, which means that these gene products may be involved yet are inadequate to promote cell survival.

Ca2+/calmodulin-dependent protein kinase II phosphorylation of the presynaptic protein synapsin I is persistently increased during long-term potentiation

Long-term potentiation (LTP) is an increase in synaptic responsiveness thought to be involved in mammalian learning and memory. The localization (presynaptic and/or postsynaptic) of changes underlying LTP has been difficult to resolve with current electrophysiological techniques. Using a biochemical approach, we have addressed this issue and attempted to identify specific molecular mechanisms that may underlie LTP. We utilized a novel multiple-electrode stimulator to produce LTP in a substantial portion of the synapses in a hippocampal CA1 minislice and tested the effects of such stimulation on the presynaptic protein synapsin I. LTP-inducing stimulation produced a long-lasting 6-fold increase in the phosphorylation of synapsin I at its Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) sites without affecting synapsin I levels. This effect was fully blocked by either the N-methyl-d-aspartate receptor antagonist d(−)-2-amino-5-phosphonopentanoic acid (APV) or the CaM kinase II inhibitor KN-62. Our results indicate that LTP expression is accompanied by persistent changes in presynaptic phosphorylation, and specifically that presynaptic CaM kinase II activity and synapsin I phosphorylation may be involved in LTP expression.

Survival function of ERK1/2 as IL-3-activated, staurosporine-resistant Bcl2 kinases

Bcl2 phosphorylation at Ser-70 may be required for the full and potent suppression of apoptosis in IL-3-dependent myeloid cells and can result from agonist activation of mitochondrial protein kinase C (PKC). Paradoxically, expression of exogenous Bcl2 can protect parental cells from apoptosis induced by the potent PKC inhibitor, staurosporine (stauro). High concentrations of stauro of up to 1 μM only partially inhibit IL-3-stimulated Bcl2 phosphorylation but completely block PKC-mediated Bcl2 phosphorylation in vitro. These data indicate a role for a stauro-resistant Bcl2 kinase (SRK). We show that aurintricarboxylic acid (ATA), a nonpeptide activator of cellular MEK/mitogen-activated protein kinase (MAPK) kinase, can induce Ser-70 phosphorylation of Bcl2 and support survival of cells expressing wild-type but not the phosphorylation-incompetent S70A mutant Bcl2. A role for a MEK/MAPK as a responsible SRK was implicated because the highly specific MEK/MAPK inhibitor, PD98059, also can only partially inhibit IL-3-induced Bcl2 phosphorylation, whereas the combination of PD98059 and stauro completely blocks phosphorylation and synergistically enhances apoptosis. p44MAPK/extracellular signal-regulated kinase 1 (ERK1) and p42 MAPK/ERK2 are activated by IL-3, colocalize with mitochondrial Bcl2, and can directly phosphorylate Bcl2 on Ser-70 in a stauro-resistant manner both in vitro and in vivo. These findings suggest a role for the ERK1/2 kinases as SRKs. Thus, the SRKs can serve to functionally link the IL-3-stimulated proliferative and survival signaling pathways and, in a novel capacity, may explain how Bcl2 can suppress stauro-induced apoptosis. In addition, although the mechanism of regulation of Bcl2 by phosphorylation is not yet clear, our results indicate that phosphorylation may functionally stabilize the Bcl2-Bax heterodimerization.

Exercise-induced changes in expression and activity of proteins involved in insulin signal transduction in skeletal muscle: Differential effects on insulin-receptor substrates 1 and 2

Level of physical activity is linked to improved glucose homeostasis. We determined whether exercise alters the expression and/or activity of proteins involved in insulin-signal transduction in skeletal muscle. Wistar rats swam 6 h per day for 1 or 5 days. Epitrochlearis muscles were excised 16 h after the last exercise bout, and were incubated with or without insulin (120 nM). Insulin-stimulated glucose transport increased 30% and 50% after 1 and 5 days of exercise, respectively. Glycogen content increased 2- and 4-fold after 1 and 5 days of exercise, with no change in glycogen synthase expression. Protein expression of the glucose transporter GLUT4 and the insulin receptor increased 2-fold after 1 day, with no further change after 5 days of exercise. Insulin-stimulated receptor tyrosine phosphorylation increased 2-fold after 5 days of exercise. Insulin-stimulated tyrosine phosphorylation of insulin-receptor substrate (IRS) 1 and associated phosphatidylinositol (PI) 3-kinase activity increased 2.5- and 3.5-fold after 1 and 5 days of exercise, despite reduced (50%) IRS-1 protein content after 5 days of exercise. After 1 day of exercise, IRS-2 protein expression increased 2.6-fold and basal and insulin-stimulated IRS-2 associated PI 3-kinase activity increased 2.8-fold and 9-fold, respectively. In contrast to IRS-1, IRS-2 expression and associated PI 3-kinase activity normalized to sedentary levels after 5 days of exercise. Insulin-stimulated Akt phosphorylation increased 5-fold after 5 days of exercise. In conclusion, increased insulin-stimulated glucose transport after exercise is not limited to increased GLUT4 expression. Exercise leads to increased expression and function of several proteins involved in insulin-signal transduction. Furthermore, the differential response of IRS-1 and IRS-2 to exercise suggests that these molecules have specialized, rather than redundant, roles in insulin signaling in skeletal muscle.

Analysis of receptor signaling pathways by mass spectrometry: Identification of Vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors

Oligomerization of receptor protein tyrosine kinases such as the epidermal growth factor receptor (EGFR) by their cognate ligands leads to activation of the receptor. Transphosphorylation of the receptor subunits is followed by the recruitment of signaling molecules containing src homology 2 (SH2) or phosphotyrosine interaction domains (PID). Additionally, several cytoplasmic proteins that may or may not associate with the receptor undergo tyrosine phosphorylation. To identify several components of the EGFR signaling pathway in a single step, we have immunoprecipitated molecules that are tyrosine phosphorylated in response to EGF and analyzed them by one-dimensional gel electrophoresis followed by mass spectrometry. Combining matrix-assisted laser desorption/ionization (MALDI) and nanoelectrospray tandem mass spectrometry (MS/MS) led to the identification of nine signaling molecules, seven of which had previously been implicated in EGFR signaling. Several of these molecules were identified from low femtomole levels of protein loaded onto the gel. We identified Vav-2, a recently discovered guanosine nucleotide exchange factor that is expressed ubiquitously, as a substrate of the EGFR. We demonstrate that Vav-2 is phosphorylated on tyrosine residues in response to EGF and associates with the EGFR in vivo. Binding of Vav-2 to the EGFR is mediated by the SH2 domain of Vav-2. In keeping with its ubiquitous expression, Vav-2 seems to be a general signaling molecule, since it also associates with the platelet-derived growth factor (PDGF) receptor and undergoes tyrosine phosphorylation in fibroblasts upon PDGF stimulation. The strategy suggested here can be used for routine identification of downstream components of cell surface receptors in mammalian cells.

Bilirubin, formed by activation of heme oxygenase-2, protects neurons against oxidative stress injury

Heme oxygenase (HO) catalyzes the conversion of heme to carbon monoxide, iron, and biliverdin, which is immediately reduced to bilirubin (BR). Two HO active isozymes exist: HO1, an inducible heat shock protein, and HO2, which is constitutive and highly concentrated in neurons. We demonstrate a neuroprotective role for BR formed from HO2. Neurotoxicity elicited by hydrogen peroxide in hippocampal and cortical neuronal cultures is prevented by the phorbol ester, phorbol 12-myristate 13-acetate (PMA) via stimulation of protein kinase C. We observe phosphorylation of HO2 through the protein kinase C pathway with enhancement of HO2 catalytic activity and accumulation of BR in neuronal cultures. The neuroprotective effects of PMA are prevented by the HO inhibitor tin protoporphyrin IX and in cultures from mice with deletion of HO2 gene. Moreover, BR, an antioxidant, is neuroprotective at nanomolar concentrations.

A role for the light-dependent phosphorylation of visual arrestin

Arrestins are regulatory proteins that participate in the termination of G protein-mediated signal transduction. The major arrestin in the Drosophila visual system, Arrestin 2 (Arr2), is phosphorylated in a light-dependent manner by a Ca2+/calmodulin-dependent protein kinase and has been shown to be essential for the termination of the visual signaling cascade in vivo. Here, we report the isolation of nine alleles of the Drosophila photoreceptor cell-specific arr2 gene. Flies carrying each of these alleles underwent light-dependent retinal degeneration and displayed electrophysiological defects typical of previously identified arrestin mutants, including an allele encoding a protein that lacks the major Ca2+/calmodulin-dependent protein kinase site. The phosphorylation mutant had very low levels of phosphorylation and lacked the light-dependent phosphorylation observed with wild-type Arr2. Interestingly, we found that the Arr2 phosphorylation mutant was still capable of binding to rhodopsin; however, it was unable to release from membranes once rhodopsin had converted back to its inactive form. This finding suggests that phosphorylation of arrestin is necessary for the release of arrestin from rhodopsin. We propose that the sequestering of arrestin to membranes is a possible mechanism for retinal disease associated with previously identified rhodopsin alleles in humans.

Biosynthesis of terpenoids: 4-Diphosphocytidyl-2-C-methyl-d-erythritol kinase from tomato

The putative catalytic domain (residues 81–401) of a predicted tomato protein with similarity to 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase of Escherichia coli was expressed in a recombinant E. coli strain. The protein was purified to homogeneity and was shown to catalyze the phosphorylation of the position 2 hydroxy group of 4-diphosphocytidyl-2-C-methyl-d-erythritol at a rate of 33 μmol⋅mg−1⋅min−1. The structure of the reaction product, 4-diphosphocytidyl-2-C-methyl-d-erythritol 2-phosphate, was established by NMR spectroscopy. Divalent metal ions, preferably Mg2+, are required for activity. Neither the tomato enzyme nor the E. coli ortholog catalyzes the phosphorylation of isopentenyl monophosphate.