THE ROLE OF TAK1 IN PANCREATIC CANCER DEVELOPMENT

Pancreatic cancer is the fourth leading cause of cancer-related mortality in the United States and the fifth leading cause of cancer-related mortality worldwide. Pancreatic cancer is a big challenge in large due to the lack of early symptoms. In addition, drug resistance is a major obstacle to the success of chemotherapy in pancreatic cancer. The underlying mechanism of drug resistance in human pancreatic cancers is not well understood. Better understanding of the mechanism of molecular pathways in human pancreatic cancers can help to identify the novel therapeutic target candidates, and develop the new preventive and clinic strategies to improve patient survival. We discovered that TAK1 is overexpressed in pancreatic cancer cell lines and patient tumor tissues. We demonstrated that the elevated activity of TAK1 is caused by its binding partner TAB1. Knocking down of TAK1 in pancreatic cancer cells with RNAi technique resulted in cell apoptosis and significantly reduces the size of tumors in mice and made a chemotherapy drug more potent. Targeting the kinase activity of TAK1 with the selective inhibitor LY2610956 strongly synergized in vitro with the antitumor activity of gemcitabine, oxaliplatin, or irinotecan on pancreatic cancer cells. These findings highlighted that TAK1 could be a potential therapeutic target for pancreatic cancer. We also demonstrated that TAK activity is regulated by its binding protein TAB1. We defined a minimum TAB1 sequence which is required and sufficient for TAK1 kinase activity. We created a recombinant TAK1-TAB1 C68 fusion form which has highly kinase activity. This active form could is used for screening TAK1 inhibitors. In addition, several posttranslational modifications were identified in our study. The acetylation of lysine 158 on TAK1 is required for kinase activity. This site is conserved throughout all of kinase. Our findings may reveal a new mechanism by which kinase activity is regulated.

Ubiquitylation of voltage-gated sodium channels.

Ion channel proteins are regulated by different types of posttranslational modifications. The focus of this review is the regulation of voltage-gated sodium channels (Navs) upon their ubiquitylation. The amiloride-sensitive epithelial sodium channel (ENaC) was the first ion channel shown to be regulated upon ubiquitylation. This modification results from the binding of ubiquitin ligase from the Nedd4 family to a protein-protein interaction domain, known as the PY motif, in the ENaC subunits. Many of the Navs have similar PY motifs, which have been demonstrated to be targets of Nedd4-dependent ubiquitylation, tagging them for internalization from the cell surface. The role of Nedd4-dependent regulation of the Nav membrane density in physiology and disease remains poorly understood. Two recent studies have provided evidence that Nedd4-2 is downregulated in dorsal root ganglion (DRG) neurons in both rat and mouse models of nerve injury-induced neuropathic pain. Using two different mouse models, one with a specific knockout of Nedd4-2 in sensory neurons and another where Nedd4-2 was overexpressed with the use of viral vectors, it was demonstrated that the neuropathy-linked neuronal hyperexcitability was the result of Nav1.7 and Nav1.8 overexpression due to Nedd4-2 downregulation. These studies provided the first in vivo evidence of the role of Nedd4-2-dependent regulation of Nav channels in a disease state. This ubiquitylation pathway may be involved in the development of symptoms and diseases linked to Nav-dependent hyperexcitability, such as pain, cardiac arrhythmias, epilepsy, migraine, and myotonias.

Expressed protein ligation: A general method for protein engineering

A protein semisynthesis method—expressed protein ligation—is described that involves the chemoselective addition of a peptide to a recombinant protein. This method was used to ligate a phosphotyrosine peptide to the C terminus of the protein tyrosine kinase C-terminal Src kinase (Csk). By intercepting a thioester generated in the recombinant protein with an N-terminal cysteine containing synthetic peptide, near quantitative chemical ligation of the peptide to the protein was achieved. The semisynthetic tail-phosphorylated Csk showed evidence of an intramolecular phosphotyrosine-Src homology 2 interaction and an unexpected increase in catalytic phosphoryl transfer efficiency toward a physiologically relevant substrate compared with the non-tail-phosphorylated control. This work illustrates that expressed protein ligation is a simple and powerful new method in protein engineering to introduce sequences of unnatural amino acids, posttranslational modifications, and biophysical probes into proteins of any size.

Structure of the Ets-1 pointed domain and mitogen-activated protein kinase phosphorylation site

The Pointed (PNT) domain and an adjacent mitogen-activated protein (MAP) kinase phosphorylation site are defined by sequence conservation among a subset of ets transcription factors and are implicated in two regulatory strategies, protein interactions and posttranslational modifications, respectively. By using NMR, we have determined the structure of a 110-residue fragment of murine Ets-1 that includes the PNT domain and MAP kinase site. The Ets-1 PNT domain forms a monomeric five-helix bundle. The architecture is distinct from that of any known DNA- or protein-binding module, including the helix-loop-helix fold proposed for the PNT domain of the ets protein TEL. The MAP kinase site is in a highly flexible region of both the unphosphorylated and phosphorylated forms of the Ets-1 fragment. Phosphorylation alters neither the structure nor monomeric state of the PNT domain. These results suggest that the Ets-1 PNT domain functions in heterotypic protein interactions and support the possibility that target recognition is coupled to structuring of the MAP kinase site.

Covalent modification of the homeodomain-interacting protein kinase 2 (HIPK2) by the ubiquitin-like protein SUMO-1

Posttranslational modifications such as ubiquitination and phosphorylation play an important role in the regulation of cellular protein function. Homeodomain-interacting protein kinase 2 (HIPK2) is a member of the recently identified family of nuclear protein kinases that act as corepressors for homeodomain transcription factors. Here, we show that HIPK2 is regulated by a ubiquitin-like protein, SUMO-1. We demonstrate that HIPK2 localizes to nuclear speckles (dots) by means of a speckle-retention signal. This speckle-retention signal contains a domain that interacts with a mouse ubiquitin-like protein conjugating (E2) enzyme, mUBC9. In cultured cells, HIPK2 is covalently modified by SUMO-1, and the SUMO-1 modification of HIPK2 correlates with its localization to nuclear speckles (dots). Thus, our results provide firm evidence that the nuclear protein kinase HIPK2 can be covalently modified by SUMO-1, which directs its localization to nuclear speckles (dots).

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.

Methylation of histone H3 at lysine 4 is highly conserved and correlates with transcriptionally active nuclei in Tetrahymena

Studies into posttranslational modifications of histones, notably acetylation, have yielded important insights into the dynamic nature of chromatin structure and its fundamental role in gene expression. The roles of other covalent histone modifications remain poorly understood. To gain further insight into histone methylation, we investigated its occurrence and pattern of site utilization in Tetrahymena, yeast, and human HeLa cells. In Tetrahymena, transcriptionally active macronuclei, but not transcriptionally inert micronuclei, contain a robust histone methyltransferase activity that is highly selective for H3. Microsequence analyses of H3 from Tetrahymena, yeast, and HeLa cells indicate that lysine 4 is a highly conserved site of methylation, which to date, is the major site detected in Tetrahymena and yeast. These data document a nonrandom pattern of H3 methylation that does not overlap with known acetylation sites in this histone. In as much as H3 methylation at lysine 4 appears to be specific to macronuclei in Tetrahymena, we suggest that this modification pattern plays a facilitatory role in the transcription process in a manner that remains to be determined. Consistent with this possibility, H3 methylation in yeast occurs preferentially in a subpopulation of H3 that is preferentially acetylated.

Rapid ATM-dependent phosphorylation of MDM2 precedes p53 accumulation in response to DNA damage

The p53 tumor-suppressor protein, a key regulator of cellular responses to genotoxic stress, is stabilized and activated after DNA damage. This process is associated with posttranslational modifications of p53, some of which are mediated by the ATM protein kinase. However, these modifications alone may not account in full for p53 stabilization. p53's stability and activity are negatively regulated by the oncoprotein MDM2, whose gene is activated by p53. Conceivably, p53 function may be modulated by modifications of MDM2 as well. We show here that after treatment of cells with ionizing radiation or a radiomimetic chemical, but not UV radiation, MDM2 is phosphorylated rapidly in an ATM-dependent manner. This phosphorylation is independent of p53 and the DNA-dependent protein kinase. Furthermore, MDM2 is directly phosphorylated by ATM in vitro. These findings suggest that in response to DNA strand breaks, ATM may promote p53 activity and stability by mediating simultaneous phosphorylation of both partners of the p53-MDM2 autoregulatory feedback loop.

Identification of an additional negative regulatory region for p53 sequence-specific DNA binding

The DNA binding activity of p53 is crucial for its tumor suppressor function and is subject to tight regulation. Previous studies revealed that the inhibitory function of the p53 C terminus is implicated in the latent, low affinity sequence-specific DNA binding activity of p53 in the uninduced state. Sequence-specific DNA binding of p53 has been shown to be activated by several posttranslational modifications and interacting proteins that target predominantly the C terminus. Moreover, several authors have shown that synthetic peptides corresponding to p53 C-terminal sequences activate p53 sequence-specific DNA binding. In an effort to identify the interaction site of p53 with these activating peptides we assessed complex formation between p53 deletion constructs and C-terminal activating peptides by peptide affinity precipitation. This study revealed that two distal regions of the p53 molecule contribute synergistically to the interaction with activating C-terminal peptides: amino acids 80–93 and 364–393. The C-terminal residues 364–393 are already well characterized as having negative regulatory function. DNA binding analyses with these deletion constructs reveal a comparable negative regulatory activity for residues 80–93, defining this region as a previously unidentified negative regulatory domain of p53. Furthermore, synthetic peptides spanning this newly identified proline-rich negative regulatory region (residues 80–93) are able to activate p53 sequence-specific DNA binding in vitro. We suggest that both negative regulatory regions, residues 80–93 and 364–393, contribute cooperatively to the maintenance of the latent, low-affinity DNA binding conformation of p53.

Cloning and mitochondrial localization of full-length D-AKAP2, a protein kinase A anchoring protein

Differential compartmentalization of signaling molecules in cells and tissues is being recognized as an important mechanism for regulating the specificity of signal transduction pathways. A kinase anchoring proteins (AKAPs) direct the subcellular localization of protein kinase A (PKA) by binding to its regulatory (R) subunits. Dual specific AKAPs (D-AKAPs) interact with both RI and RII. A 372-residue fragment of mouse D-AKAP2 with a 40-residue C-terminal PKA binding region and a putative regulator of G protein signaling (RGS) domain was previously identified by means of a yeast two-hybrid screen. Here, we report the cloning of full-length human D-AKAP2 (662 residues) with an additional putative RGS domain, and the corresponding mouse protein less the first two exons (617 residues). Expression of D-AKAP2 was characterized by using mouse tissue extracts. Full-length D-AKAP2 from various tissues shows different molecular weights, possibly because of alternative splicing or posttranslational modifications. The cloned human gene product has a molecular weight similar to one of the prominent mouse proteins. In vivo association of D-AKAP2 with PKA in mouse brain was demonstrated by using cAMP agarose pull-down assay. Subcellular localization for endogenous mouse, rat, and human D-AKAP2 was determined by immunocytochemistry, immunohistochemistry, and tissue fractionation. D-AKAP2 from all three species is highly enriched in mitochondria. The mitochondrial localization and the presence of RGS domains in D-AKAP2 may have important implications for its function in PKA and G protein signal transduction.

Subunit cleavage of mosquito pro-vitellogenin by a subtilisin-like convertase.

The eukaryotic convertase family plays an important role in posttranslational proteolytic processing and activation of many pro- and polypeptides that have at their cleavage sites the paired basic motif, RX(K/R)R. Recent studies have revealed that the cleavage site of insect pro-vitellogenins (pro-Vg) also contains this motif. To identify and characterize the insect pro-Vg processing enzyme, Vg convertase (VC), its cDNA was cloned from a vitellogenic female fat body cDNA library of the mosquito, Aedes aegypti. The 3735-bp-long VC cDNA has an open reading frame encoding a 115-kDa protein. In vitro transcription/translation of VC cDNA revealed that this 115-kDa protein becomes 140 kDa after co- and posttranslational modifications. The VC deduced amino acid sequence has high similarity to and a domain structure characteristic of furin-like convertases. Northern blot analysis showed that a single 4.2-kb transcript was expressed in the fat body during the first 18 hr of the Vg synthetic period. Coexpression of VC cDNA with mosquito Vg cDNA resulted in correct cleavage of pro-Vg. Thus, this newly identified convertase is, indeed, a functional fat body-specific enzyme for pro-Vg cleavage.

Association of a M(r) 90,000 phosphoprotein with protein kinase PKR in cells exhibiting enhanced phosphorylation of translation initiation factor eIF-2 alpha and premature shutoff of protein synthesis after infection with gamma 134.5- mutants of herpes simplex virus 1.

The protein encoded by the gamma 134.5 gene of herpes simplex virus precludes premature shutoff of protein synthesis in human cells triggered by stress associated with onset of viral DNA synthesis. The carboxyl terminus of the protein is essential for this function. This report indicates that the shutoff of protein synthesis is not due to mRNA degration because mRNA from wild-type or gamma 134.5- virus-infected cells directs protein synthesis. Analyses of the posttranslational modifications of translation initiation factor eIF-2 showed the following: (i) eIF-2 alpha was selectively phosphorylated by a kinase present in ribosome-enriched fraction of cells infected with gamma 134.5- virus. (ii) Endogenous eIF-2 alpha was totally phosphorylated in cells infected with gamma 134.5- virus or a virus lacking the 3' coding domain of the gamma 134.5 gene but was not phosphorylated in mock-infected or wild-type virus-infected cells. (iii) Immune precipitates of the PKR kinase that is responsible for regulation of protein synthesis of some cells by phosphorylation of eIF-2 alpha yielded several phosphorylated polypeptides. Of particular significance were two observations. First, phosphorylation of PKR kinase was elevated in all infected cells relative to the levels in mock-infected cells. Second, the precipitates from lysates of cells infected with gamma 134.5- virus or a virus lacking the 3' coding domain of the gamma 134.5 gene contained an additional labeled phosphoprotein of M(r) 90,000 (p90). This phosphoprotein was present in only trace amounts in the immunoprecipitate from cells infected with wild-type virus or mutants lacking a portion of the 5' domain of gamma 134.5. We conclude that in the absence of gamma 134.5 protein, PKR kinase complexes with the p90 phosphoprotein and shuts off protein synthesis by phosphorylation of the alpha subunit of translation initiation factor eIF-2.

Sphingosylphosphocholine, a signaling molecule which accumulates in Niemann-Pick disease type A, stimulates DNA-binding activity of the transcription activator protein AP-1.

Sphingosylphosphocholine (SPC) is the deacylated derivative of sphingomyelin known to accumulate in neuropathic Niemann-Pick disease type A. SPC is a potent mitogen that increases intracellular free Ca2+ and free arachidonate through pathways that are only partly protein kinase C-dependent. Here we show that SPC increased specific DNA-binding activity of transcription activator AP-1 in electrophoretic mobility-shift assays. Increased DNA-binding activity of AP-1 was detected after only 1-3 min, was maximal after 6 hr, and remained elevated at 12-24 hr. c-Fos was found to be a component of the AP-1 complex. Northern hybridization revealed an increase in c-fos transcripts after 30 min. Since the increase in AP-1 binding activity preceded the increase in c-fos mRNA, posttranslational modifications may be important in mediating the early SPC-induced increases in AP-1 DNA-binding activity. Western analysis detected increases in nuclear c-Jun and c-Fos proteins following SPC treatment. SPC also transactivated a reporter gene construct through the AP-1 recognition site, indicating that SPC can regulate the expression of target genes. Thus, SPC-induced cell proliferation may result from activation of AP-1, linking signal transduction by SPC to gene expression. Since the expression of many proteins with diverse functions is known to be regulated by AP-1, SPC-induced activation of AP-1 may contribute to the pathophysiology of Niemann-Pick disease.

Rapid mass spectrometric peptide sequencing and mass matching for characterization of human melanoma proteins isolated by two-dimensional PAGE.

We report a general mass spectrometric approach for the rapid identification and characterization of proteins isolated by preparative two-dimensional polyacrylamide gel electrophoresis. This method possesses the inherent power to detect and structurally characterize covalent modifications. Absolute sensitivities of matrix-assisted laser desorption ionization and high-energy collision-induced dissociation tandem mass spectrometry are exploited to determine the mass and sequence of subpicomole sample quantities of tryptic peptides. These data permit mass matching and sequence homology searching of computerized peptide mass and protein sequence data bases for known proteins and design of oligonucleotide probes for cloning unknown proteins. We have identified 11 proteins in lysates of human A375 melanoma cells, including: alpha-enolase, cytokeratin, stathmin, protein disulfide isomerase, tropomyosin, Cu/Zn superoxide dismutase, nucleoside diphosphate kinase A, galaptin, and triosephosphate isomerase. We have characterized several posttranslational modifications and chemical modifications that may result from electrophoresis or subsequent sample processing steps. Detection of comigrating and covalently modified proteins illustrates the necessity of peptide sequencing and the advantages of tandem mass spectrometry to reliably and unambiguously establish the identity of each protein. This technology paves the way for studies of cell-type dependent gene expression and studies of large suites of cellular proteins with unprecedented speed and rigor to provide information complementary to the ongoing Human Genome Project.

The isoflavonoids genistein and quercetin activate different stress signaling pathways as shown by analysis of site-specific phosphorylation of ATM, p53 and histone H2AX

The ataxia-telangiectasia mutated (ATM) protein kinase is activated in response to ionizing radiation (IR) and activates downstream DNA-damage signaling pathways. Although the role of ATM in the cellular response to ionizing radiation has been well characterized, its role in response to other DNA-damaging agents is less well defined. We previously showed that genistein, a naturally occurring isoflavonoid, induced increased ATM protein kinase activity, ATM-dependent phosphorylation of p53 on serine 15 and activation of the DNA-binding properties of p53. Here. we show that genistein also induces phosphorylation of p53 at serines 6, 9, 20,46, and 392, and that genistein-induced accumulation and phosphorylation of p53 is reduced in two ATM-deficient human cell lines. Also, we show that genistein induces phosphorylation of ATM on serine 1981 and phosphorylation of histone H2AX on serine 139. The related bioflavonoids, daidzein and biochanin A, did not induce either phosphorylation of p53 or ATM at these sites. Like genistein, quercetin induced phosphorylation of ATM on serine 198 1, and ATM-dependent phosphorylation of histone H2AX on serine 139; however, p53 accumulation and phosphorylation on serines 6, 9, 15, 20, 46, and 392 occurred in ATM-deficient cells, indicating that ATM is not required for quercetin-induced phosphorylation of p53. Our data suggest that genistein and quercetin induce different DNA-damage induced signaling pathways that, in the case of genistein, are highly ATM-dependent but, in the case of quercetin, may be ATM-dependent only for some downstream targets. (C) 2003 Elsevier B.V. All rights reserved.