Biomedical subjects

Philip A Cole

Publications and source records attributed to Philip A Cole.

At least 19 recordsLinked to original sources

PubMed2025

Analysis of a BCOR internal tandem duplication in mouse embryonic stem cell to neuronal precursor differentiation.

BCOR (BCL-6 corepressor) is a component of variant Polycomb Repressive Complex 1.1 (vPRC1.1), one of several vPRC1 complexes that catalyze histone H2A ubiquitination thought to play an important role in PRC2 binding and the deposition of H3K27me3 to silence genes. The PCGF Ub-like fold discriminator (PUFD) domain at the C-terminus of BCOR forms a heterodimer with PCGF1, serving as a critical interface for its polycomb-dependent functions. Internal tandem duplication (ITD) mutations in BCOR, causing in-frame duplications of 20 to 40 amino acids in the PUFD domain, are observed in heterogeneous tumors including sarcomas (kidney, bone, and endometrium) and neuroepithelial tumors in the brain. To dissect the molecular mechanisms underlying aberrant function of BCOR-ITD mutants, we employed mouse embryonic stem (mES) cells expressing either transgenic or endogenous BCOR-ITD. Our results indicate that the BCOR-ITD mutation does not disrupt the BCOR-PCGF1 interaction, instead maintaining the integrity of the vPRC1.1 complex. While displaying subtle changes in imprinted gene expression during differentiation toward a neural lineage, BCOR-ITD mutants also had no growth phenotype in culture. Furthermore, we found that CD24+ cells were enriched, as expected, during neural progenitor differentiation in both wildtype and mutant cells. However, sensitization of BCOR-ITD mES cells with EZH2 inhibitor during differentiation resulted in an unexpected enrichment of a CD24+CD26+ subpopulation, indicating aberrant cell fate that was also prevalent in a BCOR truncation mutant. Together, our results suggest that BCOR-ITD may largely retain wildtype function, but with increased susceptibility to synergistic stress on the Polycomb pathway.

Animals↗

PubMed2006

Protein tyrosine kinase-substrate interactions.

Protein tyrosine kinases (PTKs) are enzymes that catalyze the phosphorylation of tyrosyl residues. They are important in physiological and pathophysiological processes. Protein substrates of PTKs are often difficult to discern, but recently reported methods have helped to identify targets and characterize their structural interactions with kinases. A mechanism-based bisubstrate analog strategy has given X-ray crystallographic insights into how several topical PTKs, including the insulin receptor, Abl and epidermal growth factor receptor, interact with tyrosine-containing peptide substrates. These PTK co-crystal structures reveal both conserved and specialized features of recognition that probably contribute to substrate selection and the individual functions of these key enzymes.

Amino Acid Sequence↗

PubMed2006

Kinetic and mass spectrometric analysis of p300 histone acetyltransferase domain autoacetylation.

Acetylation of proteins by p300 histone acetyltransferase plays a critical role in the regulation of gene expression. The prior discovery of an autoacetylated regulatory loop in the p300 histone acetyltransferase (HAT) domain prompted us to further explore the mechanisms of p300 autoacetylation. Here we have described a kinetic and mass spectrometric analysis of p300 HAT autoacetylation. The rate of p300 HAT autoacetylation was approximately fourth order with respect to p300 HAT domain concentration and thus appeared to be a highly cooperative process. By showing that a catalytically defective p300 HAT domain could be efficiently acetylated by active p300 HAT, we deduced that autoacetylation occurs primarily by an intermolecular mechanism. This was further confirmed using a semisynthetic biotinylated p300 HAT domain that could be physically separated from the catalytically defective p300 HAT by avidin affinity chromatography. Autoacetylation catalyzed by p300 HAT was approximately 1000-fold more efficient than PCAF (p300/CREB-binding protein-associated factor)-mediated acetylation of catalytically defective p300 HAT. Using a novel tandem mass spectrometric approach, it was found to be possible to observe up to 17 autoacetylation events within the intact p300 regulatory loop. Kinetic analysis of the site specificity of p300 autoacetylation reveals a class of rapid events followed by a slower set of modifications. Several of these rapid autoacetylation sites correlate with an acetyltransferase-activating function based on prior mutagenesis analysis.

Acetylation↗

PubMed2006

Phosphoproteomic analysis of Her2/neu signaling and inhibition.

Her2/neu (Her2) is a tyrosine kinase belonging to the EGF receptor (EGFR)/ErbB family and is overexpressed in 20-30% of human breast cancers. We sought to characterize Her2 signal transduction pathways further by using MS-based quantitative proteomics. Stably transfected cell lines overexpressing Her2 or empty vector were generated, and the effect of an EGFR and Her2 selective tyrosine kinase inhibitor, PD168393, on these cells was characterized. Quantitative measurements were obtained on 462 proteins by using the SILAC (stable isotope labeling with amino acids in cell culture) method to monitor three conditions simultaneously. Of these proteins, 198 showed a significant increase in tyrosine phosphorylation in Her2-overexpressing cells, and 81 showed a significant decrease in phosphorylation. Treatment of Her2-overexpressing cells with PD168393 showed rapid reversibility of the majority of the Her2-triggered phosphorylation events. Phosphoproteins that were identified included many known Her2 signaling molecules as well as known EGFR signaling proteins that had not been previously linked to Her2, such as Stat1, Dok1, and delta-catenin. Importantly, several previously uncharacterized Her2 signaling proteins were identified, including Axl tyrosine kinase, the adaptor protein Fyb, and the calcium-binding protein Pdcd-6/Alg-2. We also identified a phosphorylation site in Her2, Y877, which is located in the activation loop of the kinase domain, is distinct from the known C-terminal tail autophosphorylation sites, and may have important implications for regulation of Her2 signaling. Network modeling, which combined phosphoproteomic results with literature-curated protein-protein interaction data, was used to suggest roles for some of the previously unidentified Her2 signaling proteins.

Amino Acid Sequence↗

PubMed2006

An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor.

The mechanism by which the epidermal growth factor receptor (EGFR) is activated upon dimerization has eluded definition. We find that the EGFR kinase domain can be activated by increasing its local concentration or by mutating a leucine (L834R) in the activation loop, the phosphorylation of which is not required for activation. This suggests that the kinase domain is intrinsically autoinhibited, and an intermolecular interaction promotes its activation. Using further mutational analysis and crystallography we demonstrate that the autoinhibited conformation of the EGFR kinase domain resembles that of Src and cyclin-dependent kinases (CDKs). EGFR activation results from the formation of an asymmetric dimer in which the C-terminal lobe of one kinase domain plays a role analogous to that of cyclin in activated CDK/cyclin complexes. The CDK/cyclin-like complex formed by two kinase domains thus explains the activation of EGFR-family receptors by homo- or heterodimerization.

Allosteric Regulation↗

PubMed2006

The role of the phospho-CDK2/cyclin A recruitment site in substrate recognition.

Phospho-CDK2/cyclin A, a kinase that is active in cell cycle S phase, contains an RXL substrate recognition site that is over 40 A from the catalytic site. The role of this recruitment site, which enhances substrate affinity and catalytic efficiency, has been investigated using peptides derived from the natural substrates, namely CDC6 and p107, and a bispeptide inhibitor in which the gamma-phosphate of ATP is covalently attached by a linker to the CDC6 substrate peptide. X-ray studies with a 30-residue CDC6 peptide in complex with pCDK2/cyclin A showed binding of a dodecamer peptide at the recruitment site and a heptapeptide at the catalytic site, but no density for the linking 11 residues. Kinetic studies established that the CDC6 peptide had an 18-fold lower Km compared with heptapeptide substrate and that this effect required the recruitment peptide to be covalently linked to the substrate peptide. X-ray studies with the CDC6 bispeptide showed binding of the dodecamer at the recruitment site and the modified ATP in two alternative conformations at the catalytic site. The CDC6 bispeptide was a potent inhibitor competitive with both ATP and peptide substrate of pCDK2/cyclin A activity against a heptapeptide substrate (Ki = 0.83 nm) but less effective against RXL-containing substrates. We discuss how localization at the recruitment site (KD 0.4 microm) leads to increased catalytic efficiency and the design of a potent inhibitor. The notion of a flexible linker between the sites, which must have more than a minimal number of residues, provides an explanation for recognition and discrimination against different substrates.

Adenosine Triphosphate↗

PubMed2006

A Src-like inactive conformation in the abl tyrosine kinase domain.

The improper activation of the Abl tyrosine kinase results in chronic myeloid leukemia (CML). The recognition of an inactive conformation of Abl, in which a catalytically important Asp-Phe-Gly (DFG) motif is flipped by approximately 180 degrees with respect to the active conformation, underlies the specificity of the cancer drug imatinib, which is used to treat CML. The DFG motif is not flipped in crystal structures of inactive forms of the closely related Src kinases, and imatinib does not inhibit c-Src. We present a structure of the kinase domain of Abl, determined in complex with an ATP-peptide conjugate, in which the protein adopts an inactive conformation that resembles closely that of the Src kinases. An interesting aspect of the Src-like inactive structure, suggested by molecular dynamics simulations and additional crystal structures, is the presence of features that might facilitate the flip of the DFG motif by providing room for the phenylalanine to move and by coordinating the aspartate side chain as it leaves the active site. One class of mutations in BCR-Abl that confers resistance to imatinib appears more likely to destabilize the inactive Src-like conformation than the active or imatinib-bound conformations. Our results suggest that interconversion between distinctly different inactive conformations is a characteristic feature of the Abl kinase domain.

Amino Acid Motifs↗

PubMed2006

A mechanism-based inactivator for histone demethylase LSD1.

Histone demethylase LSD1 is a flavin-dependent amine oxidase that catalyzes the oxidative removal of one or two methyl groups from the methyl-lysine-4 side chain of histone H3. We have designed and synthesized two peptide-based inhibitor analogues that block LSD1. One of these inhibitors, compound 1, contains a propargylamine functionality and shows time-dependent inactivation of LSD1. Peptide substrate, diMeK4H3-21, protected LSD1 against inactivation by 1 in a concentration-dependent fashion. Mass spectrometric analysis showed that 1 forms a covalent interaction with FAD. Compound 1 did not detectably inhibit monoamine oxidase B in the concentration range studied. Compound 1 is thus a selective, mechanism-based inactivator of LSD1 and is likely to serve as a useful tool in the study of histone modifications and chromatin remodeling.

Amino Acid Sequence↗

PubMed2006

Negative regulation of a protein tyrosine phosphatase by tyrosine phosphorylation.

The low molecular weight protein tyrosine phosphatase (LMW-PTP) is a ubiquitously expressed enzyme with several proposed roles in cell signaling. Previously, two tyrosine phosphorylation modifications of LMW-PTP at sites Tyr-131 and Tyr-132 in response to growth factor stimulation have been mapped and suggested to stimulate LMW-PTP phosphatase activity. Biochemical analysis of tyrosine phosphorylation of a tyrosine phosphatase is challenging because of the intrinsic instability of these modifications. Here we used expressed protein ligation to site-specifically incorporate a phosphotyrosine mimic (phosphonomethylenephenylalanine, Pmp) at the Tyr-131 and Tyr-132 positions and measured the catalytic activity of these semisynthetic LMW-PTPs. The phosphonate-modified LMW-PTPs were 10- to 23-fold less active in dephosphorylating phosphotyrosine peptides derived from the PDGF receptor and p190RhoGap, two putative cellular substrates. These findings suggest the first example of a tyrosine phosphatase that is inhibited by tyrosine phosphorylation and provide a new model for the regulation of LMW-PTP and its role in cell adhesion.

Amino Acid Sequence↗

PubMed2006

Chemical rescue of a mutant enzyme in living cells.

The restoration of catalytic activity to mutant enzymes by small molecules is well established for in vitro systems. Here, we show that the protein tyrosine kinase Src arginine-388-->alanine (R388A) mutant can be rescued in live cells with the use of the small molecule imidazole. Cellular rescue of a viral Src homolog was rapid and reversible and conferred predicted oncogenic properties. Using chemical rescue in combination with mass spectrometry, we confirmed six known Src kinase substrates and identified several new protein targets. Chemical rescue data suggest that cellular Src is active under basal conditions. Rescue of R388A cellular Src provided insights into the mitogen-activated protein kinase pathway. This chemical rescue approach will likely have many applications in cell signaling.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Synthesis and evaluation of a potent and selective cell-permeable p300 histone acetyltransferase inhibitor.

This paper describes the first potent and selective p300 histone acetyltransferase (HAT) inhibitor which is effective in live cells. This compound 7 is a coenzyme A analogue conjugated to a cell permeabilizing oligoArg peptide via disulfide linkage. This compound was shown to block cellular histone acetylation and transcription using a p300-sensitive reporter. It should thus be broadly useful for dissecting the role of p300 HAT activity in physiologic and disease states.

Journal Article↗

PubMed2005

Protein semisynthesis and expressed protein ligation: chasing a protein's tail.

The adaptation of native chemical ligation to protein semisynthesis has become a powerful way to address problems in the analysis of protein structure and function. In particular, the exploitation of nature's inteins in expressed protein ligation is now a standard approach in the study of proteins. Site-specific incorporation of unnatural amino acids, biophysical probes and post-translational modifications in proteins have led to new insights into enzyme mechanisms, protein folding, ion channel function, translation and signaling.

Amino Acids↗

PubMed2005

Protein kinase structure and function analysis with chemical tools.

Protein kinases are the largest enzyme superfamily involved in cell signal transduction and represent therapeutic targets for a range of diseases. There have been intensive efforts from many labs to understand their catalytic mechanisms, discover inhibitors and discern their cellular functions. In this review, we will describe two approaches developed to analyze protein kinases: bisubstrate analog inhibition and phosphonate analog utilization. Both of these methods have been used in combination with the protein semisynthesis method expressed protein ligation to advance our understanding of kinase-substrate interactions and functional elucidation of phosphorylation. Previous work on the nature of the protein kinase mechanism suggests it follows a dissociative transition state. A bisubstrate analog was designed against the insulin receptor kinase to mimic the geometry of a dissociative transition state reaction coordinate distance. This bisubstrate compound proved to be a potent inhibitor against the insulin receptor kinase and occupied both peptide and nucleotide binding sites. Bisubstrate compounds with altered hydrogen bonding potential as well as varying spacers between the adenine and the peptide demonstrate the importance of the original design features. We have also shown that related bisubstrate analogs can be used to potently block serine/threonine kinases including protein kinase A. Since many protein kinases recognize folded protein substrates for efficient phosphorylation, it was advantageous to incorporate the peptide-ATP conjugates into protein structures. Using expressed protein ligation, a Src-ATP conjugate was produced and shown to be a high affinity ligand for the Csk tyrosine kinase. Nonhydrolyzable mimics of phosphoSer/phosphoTyr can be useful in examining the functionality of phosphorylation events. Using expressed protein ligation, we have employed phosphonomethylene phenylalanine and phosphonomethylene alanine to probe the phosphorylation of Tyr and Ser, respectively. These tools have permitted an analysis of the SH2-phosphatases (SHP1 and SHP2), revealing a novel intramolecular stimulation of catalytic activity mediated by the corresponding phosphorylation events. They have also been used to characterize the cellular regulation of the melatonin rhythm enzyme by phosphorylation.

Animals↗

PubMed2005

Fluorescence analysis of a dynamic loop in the PCAF/GCN5 histone acetyltransferase.

PCAF and GCN5 are histone acetyltransferase (HAT) paralogs which play roles in the remodeling of chromatin in health and disease. Previously, a conformationally flexible loop in the catalytic domain had been observed in the X-ray structures of GCN5 in different liganded states. Here, the conformation and dynamics of this PCAF/GCN5 alpha5-beta6 loop was investigated in solution using tryptophan fluorescence. A mutant human PCAF HAT domain (PCAF(Wloop)) was created in which the natural tryptophan (Trp-514) remote from the alpha5-beta6 loop was replaced with tyrosine and a glutamate within the loop (Glu-641) was substituted with tryptophan. This PCAF(Wloop) protein exhibited catalytic parameters within 3-fold of those of the wild-type PCAF catalytic domain, suggesting that the loop mutation was not deleterious for HAT activity. While saturating CoASH induced a 30% quenching of Trp fluorescence in PCAF(Wloop), binding of the high-affinity bisubstrate analogue H3-CoA-20 led to a 2-fold fluorescence increase. These different effects correlate with the different alpha5-beta6 loop conformations seen previously in X-ray structures. On the basis of stopped-flow fluorescence studies, binding of H3-CoA-20 to PCAF(Wloop) proceeds via a rapid association step followed by a slower conformational change involving loop movement. Time-resolved fluorescence measurements support a model in which the alpha5-beta6 loop in the H3-CoA-20-PCAF(Wloop) complex exists in a narrower ensemble of conformations compared to free PCAF(Wloop). The relevance of loop dynamics to PCAF/GCN5 catalysis and substrate specificity are discussed.

Animals↗

PubMed2005

p300/CBP-associated factor drives DEK into interchromatin granule clusters.

DEK is a mammalian protein that has been implicated in the pathogenesis of autoimmune diseases and cancer, including acute myeloid leukemia, melanoma, glioblastoma, hepatocellular carcinoma, and bladder cancer. In addition, DEK appears to participate in multiple cellular processes, including transcriptional repression, mRNA processing, and chromatin remodeling. Sub-nuclear distribution of this protein, with the attendant functional ramifications, has remained a controversial topic. Here we report that DEK undergoes acetylation in vivo at lysine residues within the first 70 N-terminal amino acids. Acetylation of DEK decreases its affinity for DNA elements within the promoter, which is consistent with the involvement of DEK in transcriptional repression. Furthermore, deacetylase inhibition results in accumulation of DEK within interchromatin granule clusters (IGCs), sub-nuclear structures that contain RNA processing factors. Overexpression of P/CAF acetylase drives DEK into IGCs, and addition of a newly developed, synthetic, cell-permeable P/CAF inhibitor blocks this movement. To our knowledge, this is the first reported example of acetylation playing a direct role in relocation of a protein to IGCs, and this may explain how DEK can function in multiple pathways that take place in distinct sub-nuclear compartments. These findings also suggest that DEK-associated malignancies and autoimmune diseases might be amenable to treatment with agents that alter acetylation.

Acetylation↗

PubMed2005

Bisubstrate analog probes for the insulin receptor protein tyrosine kinase: molecular yardsticks for analyzing catalytic mechanism and inhibitor design.

Bisubstrate analogs have the potential to provide enhanced specificity for protein kinase inhibition and tools to understand catalytic mechanism. Previous efforts led to the design of a peptide-ATP conjugate bisubstrate analog utilizing aminophenylalanine in place of tyrosine and a thioacetyl linker to the gamma-phosphate of ATP which was a potent inhibitor of the insulin receptor kinase (IRK). In this study, we have examined the contributions of various electrostatic and structural elements in the bisubstrate analog to IRK binding affinity. Three types of changes (seven specific analogs in all) were introduced: a Tyr isostere of the previous aminophenylalanine moiety, modifications of the spacer between the adenine and the peptide, and deletions and substitutions within the peptide moiety. These studies allowed a direct evaluation of the hydrogen bond strength between the anilino nitrogen of the bisubstrate analog and the enzyme catalytic base Asp and showed that it contributes 2.5 kcal/mol of binding energy, in good agreement with previous predictions. Modifications of the linker length resulted in weakened inhibitory affinity, consistent with the geometric requirements of an enzyme-catalyzed dissociative transition state. Alterations in the peptide motif generally led to diminished inhibitory potency, and only some of these effects could be rationalized based on prior kinetic and structural studies. Taken together, these results suggest that a combination of mechanism-based design and empirical synthetic manipulation will be necessary in producing optimized protein kinase bisubstrate analog inhibitors.

Amino Acid Sequence↗

PubMed2005

Altered pharmacology and distinct coactivator usage for estrogen receptor-dependent transcription through activating protein-1.

Estrogen signaling occurs through at least two distinct molecular pathways: (i) direct binding of liganded estrogen receptors (ERs) to estrogen-responsive DNA elements (EREs) (the "ER/ERE pathway") and (ii) indirect recruitment of liganded ERs to activating protein-1 (AP-1)-responsive DNA elements via heterodimers of Fos and Jun (the "ER/AP-1 pathway"). We have developed a biochemical assay for examining ligand-regulated transcription by ERs in the ER/AP-1 pathway. This assay recapitulates the altered (i.e., agonistic) pharmacology of selective estrogen receptor modulator drugs in this pathway reported previously by using various cell-based assays. We used our biochemical assay to examine the detailed mechanisms of ER/AP-1-dependent transcription. Our studies indicate that (i) ERalpha/AP-1 complexes play a critical role in promoting the formation of stable RNA polymerase II preinitiation complexes leading to transcription initiation, (ii) chromatin is a key determinant of estrogen and selective estrogen receptor modulator signaling in the ERalpha/AP-1 pathway, (iii) distinct domains of ERalpha are required for recruitment to DNA-bound Fos/Jun heterodimers and transcriptional activation at AP-1 sites, and (iv) different enhancer/activator combinations in the ERalpha and AP-1 pathways use coactivators in distinct ways. These studies have increased our understanding of the molecular mechanisms underlying ligand-dependent signaling in the ER/AP-1 pathway and demonstrate the usefulness of this biochemical approach.

Chromatin↗

PubMed2005

Cellular stability of serotonin N-acetyltransferase conferred by phosphonodifluoromethylene alanine (Pfa) substitution for Ser-205.

Large changes in the activity of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) in the pineal gland control the rhythmic production of the time-keeping hormone melatonin. The activity of AANAT reflects changes in the amount and activation state of the AANAT protein, both of which increase at night. The molecular basis of this regulation is now becoming known, and recent data indicate that this involves phosphorylation-dependent binding to the 14-3-3 protein at two sites, one of which, Ser-205, is located several residues from the C terminus. In this study, we determined whether substitution of this residue with a non-hydrolyzable the phosphoserine/phosphothreonine mimetic would promote binding to the 14-3-3 protein and enhance cellular stability. To accomplish this, a C-terminal AANAT peptide containing the phosphonodifluoromethylene alanine at Ser-205 was synthesized and fused to bacterially expressed AANAT(30-199) using expressed protein ligation. The resulting semisynthetic protein has enhanced affinity for the expressed 14-3-3 protein and exhibits greater cellular stability in microinjection experiments, as compared with the unmodified AANAT. Enhanced 14-3-3 binding was also observed using humanized ovine AANAT, which has a different C-terminal sequence (Gly-Cys) than the ovine enzyme (Asp-Arg), indicating that that characteristic is not unique to the ovine enzyme. These studies provide the first evidence that substitution of Ser-205 with the stable phosphomimetic amino acid phosphonodifluoromethylene alanine enhances binding to 14-3-3 and the cellular stability of AANAT and are consistent with the view that Ser-205 phosphorylation plays a critical role in the regulation of AANAT activity and melatonin production.

14-3-3 Proteins↗