hMutSα- and hMutLα-dependent phosphorylation of p53 in response to DNA methylator damage

hMSH2⋅hMSH6 heterodimer (hMutSα) and hMLH1⋅hPMS2 complex (hMutLα) have been implicated in the cytotoxic response of mammalian cells to a number of DNA-damaging compounds, including methylating agents that produce O6-methylguanine (O6MeG) adducts. This study demonstrates that O6MeG lesions, in which the damaged base is paired with either T or C, are subject to excision repair in a reaction that depends on a functional mismatch repair system. Furthermore, treatment of human cells with the SN1 DNA methylators N-methyl-N-nitrosourea or N-methyl-N′-nitro-N-nitrosoguanidine results in p53 phosphorylation on serine residues 15 and 392, and these phosphorylation events depend on the presence of functional hMutSα and hMutLα. Coupled with the previous demonstration that O6MeG⋅T and O6MeG⋅C pairs are recognized by hMutSα, these results implicate action of the mismatch repair system in the initial step of a damage-signaling cascade that can lead to cell-cycle checkpoint activation or cell death in response to DNA methylator damage.

Kinase domain of the muscle-specific receptor tyrosine kinase (MuSK) is sufficient for phosphorylation but not clustering of acetylcholine receptors: Required role for the MuSK ectodomain?

Formation of the neuromuscular junction (NMJ) depends upon a nerve-derived protein, agrin, acting by means of a muscle-specific receptor tyrosine kinase, MuSK, as well as a required accessory receptor protein known as MASC. We report that MuSK does not merely play a structural role by demonstrating that MuSK kinase activity is required for inducing acetylcholine receptor (AChR) clustering. We also show that MuSK is necessary, and that MuSK kinase domain activation is sufficient, to mediate a key early event in NMJ formation—phosphorylation of the AChR. However, MuSK kinase domain activation and the resulting AChR phosphorylation are not sufficient for AChR clustering; thus we show that the MuSK ectodomain is also required. These results indicate that AChR phosphorylation is not the sole trigger of the clustering process. Moreover, our results suggest that, unlike the ectodomain of all other receptor tyrosine kinases, the MuSK ectodomain plays a required role in addition to simply mediating ligand binding and receptor dimerization, perhaps by helping to recruit NMJ components to a MuSK-based scaffold.

Phosphorylation of insulin receptor substrate 1 by glycogen synthase kinase 3 impairs insulin action

The phosphorylation of insulin receptor substrate 1 (IRS-1) on tyrosine residues by the insulin receptor (IR) tyrosine kinase is involved in most of the biological responses of insulin. IRS-1 mediates insulin signaling by recruiting SH2 proteins through its multiple tyrosine phosphorylation sites. The phosphorylation of IRS-1 on serine/threonine residues also occurs in cells; however, the particular protein kinase(s) promoting this type of phosphorylation are unknown. Here we report that glycogen synthase kinase 3 (GSK-3) is capable of phosphorylating IRS-1 and that this modification converts IRS-1 into an inhibitor of IR tyrosine kinase activity in vitro. Expression of wild-type GSK-3 or an “unregulated” mutant of the kinase (S9A) in CHO cells overexpressing IRS-1 and IR, resulted in increased serine phosphorylation levels of IRS-1, suggesting that IRS-1 is a cellular target of GSK-3. Furthermore, insulin-induced tyrosine phosphorylation of IRS-1 and IR was markedly suppressed in cells expressing wild-type or the S9A mutant, indicating that expression of GSK-3 impairs IR tyrosine kinase activity. Taken together, our studies suggest a new role for GSK-3 in attenuating insulin signaling via its phosphorylation of IRS-1 and may provide new insight into mechanisms important in insulin resistance.

Subunit rotation in Escherichia coli FoF1–ATP synthase during oxidative phosphorylation

We report evidence for proton-driven subunit rotation in membrane-bound FoF1–ATP synthase during oxidative phosphorylation. A βD380C/γC87 crosslinked hybrid F1 having epitope-tagged βD380C subunits (βflag) exclusively in the two noncrosslinked positions was bound to Fo in F1-depleted membranes. After reduction of the β–γ crosslink, a brief exposure to conditions for ATP synthesis followed by reoxidation resulted in a significant amount of βflag appearing in the β–γ crosslinked product. Such a reorientation of γC87 relative to the three β subunits can only occur through subunit rotation. Rotation was inhibited when proton transport through Fo was blocked or when ADP and Pi were omitted. These results establish FoF1 as the second example in nature where proton transport is coupled to subunit rotation.

Activation of protein kinase C by tyrosine phosphorylation in response to H2O2

Protein kinase C (PKC) isoforms, α, βI, and γ of cPKC subgroup, δ and ɛ of nPKC subgroup, and ζ of aPKC subgroup, were tyrosine phosphorylated in COS-7 cells in response to H2O2. These isoforms isolated from the H2O2-treated cells showed enhanced enzyme activity to various extents. The enzymes, PKC α and δ, recovered from the cells were independent of lipid cofactors for their catalytic activity. Analysis of mutated molecules of PKC δ showed that tyrosine residues, which are conserved in the catalytic domain of the PKC family, are critical for PKC activation induced by H2O2. These results suggest that PKC isoforms can be activated through tyrosine phosphorylation in a manner unrelated to receptor-coupled hydrolysis of inositol phospholipids.

Transforming growth factor β-induced phosphorylation of Smad3 is required for growth inhibition and transcriptional induction in epithelial cells

Drosophila Mad proteins are intracellular signal transducers of decapentaplegic (dpp), the Drosophila transforming growth factor β (TGF-β)/bone morphogenic protein (BMP) homolog. Studies in which the mammalian Smad homologs were transiently overexpressed in cultured cells have implicated Smad2 in TGF-β signaling, but the physiological relevance of the Smad3 protein in signaling by TGF-β receptors has not been established. Here we stably expressed Smad proteins at controlled levels in epithelial cells using a novel approach that combines highly efficient retroviral gene transfer and quantitative cell sorting. We show that upon TGF-β treatment Smad3 becomes rapidly phosphorylated at the SSVS motif at its very C terminus. Either attachment of an epitope tag to the C terminus or replacement of these three serine residues with alanine abolishes TGF-β-induced Smad3 phosphorylation; these proteins act in a dominant-negative fashion to block the antiproliferative effect of TGF-β in mink lung epithelial cells. A Smad3 protein in which the three C-terminal serines have been replaced by aspartic acids is also a dominant inhibitor of TGF-β signaling, but can activate plasminogen activator inhibitor 1 (PAI-1) transcription in a ligand-independent fashion when its nuclear localization is forced by transient overexpression. Phosphorylation of the three C-terminal serine residues of Smad3 by an activated TGF-β receptor complex is an essential step in signal transduction by TGF-β for both inhibition of cell proliferation and activation of the PAI-1 promoter.

Hypo-phosphorylation of the retinoblastoma protein (pRb) by cyclin D:Cdk4/6 complexes results in active pRb

In cycling cells, the retinoblastoma protein (pRb) is un- and/or hypo-phosphorylated in early G1 and becomes hyper-phosphorylated in late G1. The role of hypo-phosphorylation and identity of the relevant kinase(s) remains unknown. We show here that hypo-phosphorylated pRb associates with E2F in vivo and is therefore active. Increasing the intracellular concentration of the Cdk4/6 specific inhibitor p15INK4b by transforming growth factor β treatment of keratinocytes results in G1 arrest and loss of hypo-phosphorylated pRb with an increase in unphosphorylated pRb. Conversely, p15INK4b-independent transforming growth factor β-mediated G1 arrest of hepatocellular carcinoma cells results in loss of Cdk2 kinase activity with continued Cdk6 kinase activity and pRb remains only hypo-phosphorylated. Introduction of the Cdk4/6 inhibitor p16INK4a protein into cells by fusion to a protein transduction domain also prevents pRb hypo-phosphorylation with an increase in unphosphorylated pRb. We conclude that cyclin D:Cdk4/6 complexes hypo-phosphorylate pRb in early G1 allowing continued E2F binding.

Interleukin 3-dependent survival by the Akt protein kinase

Interleukin 3 (IL-3)-dependent survival of hematopoietic cells is known to rely on the activity of multiple signaling pathways, including a pathway leading to activation of phosphoinositide 3-kinase (PI 3-kinase), and protein kinase Akt is a direct target of PI 3-kinase. We find that Akt kinase activity is rapidly induced by the cytokine IL-3, suggesting a role for Akt in PI 3-kinase-dependent signaling in hematopoetic cells. Dominant-negative mutants of Akt specifically block Akt activation by IL-3 and interfere with IL-3-dependent proliferation. Overexpression of Akt or oncogenic v-akt protects 32D cells from apoptosis induced by IL-3 withdrawal. Apoptosis after IL-3 withdrawal is accelerated by expression of dominant-negative mutants of Akt, indicating that a functional Akt signaling pathway is necessary for cell survival mediated by the cytokine IL-3. Thus Akt appears to be an important mediator of anti-apoptotic signaling in this system.

Role of tyrosine phosphorylation of a cellular protein in adeno-associated virus 2-mediated transgene expression

The adeno-associated virus 2 (AAV), a single-stranded DNA-containing, nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. However, the single-stranded nature of the viral genome significantly impacts upon the transduction efficiency, because the second-strand viral DNA synthesis is the rate-limiting step. We hypothesized that a host-cell protein interacts with the single-stranded D sequence within the inverted terminal repeat structure of the AAV genome and prevents the viral second-strand DNA synthesis. Indeed, a cellular protein has been identified that interacts specifically and preferentially with the D sequence at the 3′ end of the AAV genome. This protein, designated the single-stranded D-sequence-binding protein (ssD-BP), is phosphorylated at tyrosine residues and blocks AAV-mediated transgene expression in infected cells by inhibiting the leading strand viral DNA synthesis. Inhibition of cellular protein tyrosine kinases by genistein results in dephosphorylation of the ssD-BP, leading not only to significant augmentation of transgene expression from recombinant AAV but also to autonomous replication of the wild-type AAV genome. Dephosphorylation of the ssD-BP also correlates with adenovirus infection, or expression of the adenovirus E4orf6 protein, which is known to induce AAV DNA replication and gene expression. Thus, phosphorylation state of the ssD-BP appears to play a crucial role in the life cycle of AAV and may prove to be an important determinant in the successful use of AAV-based vectors in human gene therapy.

Phosphorylation of two regulatory tyrosine residues in the activation of Bruton’s tyrosine kinase via alternative receptors

Mutation of Bruton’s tyrosine kinase (Btk) impairs B cell maturation and function and results in a clinical phenotype of X-linked agammaglobulinemia. Activation of Btk correlates with an increase in the phosphorylation of two regulatory Btk tyrosine residues. Y551 (site 1) within the Src homology type 1 (SH1) domain is transphosphorylated by the Src family tyrosine kinases. Y223 (site 2) is an autophosphorylation site within the Btk SH3 domain. Polyclonal, phosphopeptide-specific antibodies were developed to evaluate the phosphorylation of Btk sites 1 and 2. Crosslinking of the B cell antigen receptor (BCR) or the mast cell Fcɛ receptor, or interleukin 5 receptor stimulation each induced rapid phosphorylation at Btk sites 1 and 2 in a tightly coupled manner. Btk molecules were singly and doubly tyrosine-phosphorylated. Phosphorylated Btk comprised only a small fraction (≤5%) of the total pool of Btk molecules in the BCR-activated B cells. Increased dosage of Lyn in B cells augmented BCR-induced phosphorylation at both sites. Kinetic analysis supports a sequential activation mechanism in which individual Btk molecules undergo serial transphosphorylation (site 1) then autophosphorylation (site 2), followed by successive dephosphorylation of site 1 then site 2. The phosphorylation of conserved tyrosine residues within structurally related Tec family kinases is likely to regulate their activation.

The Fanconi anemia pathway requires FAA phosphorylation and FAA/FAC nuclear accumulation

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least eight complementation groups (A–H). Two FA genes, corresponding to complementation groups A and C, have been cloned, but the function of the FAA and FAC proteins remains unknown. We have recently shown that the FAA and FAC proteins bind and form a nuclear complex. In the current study, we analyzed the FAA and FAC proteins in normal lymphoblasts and lymphoblasts from multiple FA complementation groups. In contrast to normal controls, FA cells derived from groups A, B, C, E, F, G, and H were defective in the formation of the FAA/FAC protein complex, the phosphorylation of the FAA protein, and the accumulation of the FAA/FAC protein complex in the nucleus. These biochemical events seem to define a signaling pathway required for the maintenance of genomic stability and normal hematopoiesis. Our results support the idea that multiple gene products cooperate in the FA Pathway.

Phosphorylation of Ser-20 mediates stabilization of human p53 in response to DNA damage

Stabilization of p53 in response to DNA damage is caused by its dissociation from Mdm2, a protein that targets p53 for degradation in the proteasome. Dissociation of p53 from Mdm2 could be caused by DNA damage-induced p53 posttranslational modifications. The ATM and ATR kinases, whose activation in response to ionizing radiation (IR) and UV light, respectively, is required for p53 stabilization, directly phosphorylate p53 on Ser-15. However, phosphorylation of Ser-15 is critical for the apoptotic activity of p53 and not for p53 stabilization. Thus, whether any p53 modifications, and which, underlie disruption of the p53–Mdm2 complex after DNA damage remains to be determined. We analyzed the IR- and UV light-induced stabilization of p53 proteins with substitutions of Ser known to be posttranslationally modified after DNA damage. Substitution of Ser-20 was sufficient to abrogate p53 stabilization in response to both IR and UV light. Furthermore, both IR and UV light induced phosphorylation of p53 on Ser-20, which involved the majority of nuclear p53 protein and weakened the interaction of p53 with Mdm2 in vitro. ATM and ATR cannot phosphorylate p53 on Ser-20. We therefore propose that ATM and ATR activate an, as yet unidentified, kinase that stabilizes p53 by phosphorylating it on Ser-20.

Stress-induced phosphorylation of STAT1 at Ser727 requires p38 mitogen-activated protein kinase whereas IFN-γ uses a different signaling pathway

STAT1 is an essential transcription factor for macrophage activation by IFN-γ and requires phosphorylation of the C-terminal Ser727 for transcriptional activity. In macrophages, Ser727 phosphorylation in response to bacterial lipopolysaccharide (LPS), UV irradiation, or TNF-α occurred through a signaling path sensitive to the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580 whereas IFN-γ-mediated Ser727 phosphorylation was not inhibited by the drug. Consistently, SB203580 did not affect IFN-γ-mediated, Stat1-dependent transcription but inhibited its enhancement by LPS. Furthermore, LPS, UV irradiation, and TNF-α caused activation of p38 MAPK whereas IFN-γ did not. An essential role for p38 MAPK activity in STAT1 Ser727 phosphorylation was confirmed by using cells expressing an SB203580-resistant p38 MAPK. In such cells, STAT1 Ser727 phosphorylation in response to UV irradiation was found to be SB203580 insensitive. Targeted disruption of the mapkap-k2 gene, encoding a kinase downstream of p38 MAPK with a key role in LPS-stimulated TNF-α production and stress-induced heat shock protein 25 phosphorylation, was without a significant effect on UV-mediated Ser727 phosphorylation. The recombinant Stat1 C terminus was phosphorylated in vitro by p38MAPKα and β but not by MAPK-activated protein kinase 2. Janus kinase 2 activity, previously reported to be required for IFN-γ-mediated Ser727 phosphorylation, was not needed for LPS-mediated Ser727 phosphorylation, and activation of Janus kinase 2 did not cause the appearance of STAT1 Ser727 kinase activity. Our data suggest that STAT1 is phosphorylated at Ser727 by a stress-activated signaling pathway either through p38 MAPK directly or through an unidentified kinase downstream of p38MAPK.

The phosphorylation state of the FIGQY tyrosine of neurofascin determines ankyrin-binding activity and patterns of cell segregation

Cell–cell recognition and patterning of cell contacts have a critical role in mediating reversible assembly of a variety of transcellular complexes in the nervous system. This study provides evidence for regulation of cell interactions through modulation of ankyrin binding to neurofascin, a member of the L1CAM family of nervous system cell adhesion molecules. The phosphorylation state of the conserved FIGQY tyrosine in the cytoplasmic domain of neurofascin regulates ankyrin binding and governs neurofascin-dependent cell aggregation as well as cell sorting when neurofascin is expressed in neuroblastoma cells. These findings suggest a general mechanism for the patterning of cell contact based on external signals that regulate tyrosine phosphorylation of L1CAM members and modulate their binding to ankyrin.

CD19 and CD22 expression reciprocally regulates tyrosine phosphorylation of Vav protein during B lymphocyte signaling

B cell development and humoral immune responses are controlled by signaling thresholds established through the B lymphocyte antigen receptor (BCR) complex. BCR signaling thresholds are differentially regulated by the CD22 and CD19 cell surface receptors in vivo. B cells from CD22-deficient mice exhibit characteristics of chronic stimulation and are hyper-responsive to BCR crosslinking with augmented intracellular Ca2+ responses. By contrast, B cells from CD19-deficient mice are hypo-responsive to transmembrane signals. To identify signaling molecules involved in the positive and negative regulation of signaling thresholds, the signal transduction pathways activated after BCR crosslinking were examined in CD22- and CD19-deficient B cells. These comparisons revealed that tyrosine phosphorylation of Vav protein was uniquely augmented after BCR or CD19 crosslinking in CD22-deficient B cells, yet was modest and transient after BCR crosslinking in CD19-deficient B cells. Ligation of CD19 and CD22 in vivo is likely to positively and negatively regulate BCR signaling, respectively, because CD19 crosslinking was more efficient than BCR crosslinking at inducing Vav phosphorylation. However, simultaneous crosslinking of CD19 with the BCR resulted in a substantial decrease in Vav phosphorylation when CD22 was expressed. Thus, the differential regulation of Vav tyrosine phosphorylation by CD19 and CD22 may provide a molecular mechanism for adjusting BCR signaling thresholds.