PI3Kα Translocation Mediates Nuclear PtdIns(3,4,5)P3 Effector Signaling in Colorectal Cancer.

The canonical view of PI3Kα signaling describes phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) generation and activation of downstream effectors at the plasma membrane or at microtubule-bound endosomes. Here, we show that colorectal cancer (CRC) cell lines exhibit a diverse plasma membrane-nuclear distribution of PI3Kα, controlling corresponding levels of subcellular PtdIns(3,4,5)P3 pools. PI3Kα nuclear translocation was mediated by the importin ß-dependent nuclear import pathway. By PtdIns(3,4,5)P3 affinity capture mass spectrometry done in the presence of SDS on CRC cell lines with PI3Kα nuclear localization, we identified 867 potential nuclear PtdIns(3,4,5)P3 effector proteins. Nuclear PtdIns(3,4,5)P3 interactome proteins were characterized by noncanonical PtdIns(3,4,5)P3-binding domains and showed overrepresentation for nuclear membrane, nucleolus, and nuclear speckles. The nuclear PtdIns(3,4,5)P3 interactome was enriched for proteins related to RNA metabolism, with splicing reporter assays and SC-35 foci staining suggesting a role of epidermal growth factor-stimulated nuclear PI3Kα signaling in modulating pre-mRNA splicing. In patient tumors, nuclear p110α staining was associated with lower T stage and mucinous histology. These results indicate that PI3Kα translocation mediates nuclear PtdIns(3,4,5)P3 effector signaling in human CRC, modulating signaling responses.

Development of monoclonal anti-PDGF-CC antibodies as tools for investigating human tissue expression and for blocking PDGF-CC induced PDGFRα signalling in vivo.

PDGF-CC is a member of the platelet-derived growth factor (PDGF) family that stimulates PDGFRα phosphorylation and thereby activates intracellular signalling events essential for development but also in cancer, fibrosis and neuropathologies involving blood-brain barrier (BBB) disruption. In order to elucidate the biological and pathological role(s) of PDGF-CC signalling, we have generated high affinity neutralizing monoclonal antibodies (mAbs) recognizing human PDGF-CC. We determined the complementarity determining regions (CDRs) of the selected clones, and mapped the binding epitope for clone 6B3. Using the monoclonal 6B3, we determined the expression pattern for PDGF-CC in different human primary tumours and control tissues, and explored its ability to neutralize PDGF-CC-induced phosphorylation of PDGFRα. In addition, we showed that PDGF-CC induced disruption of the blood-retinal barrier (BRB) was significantly reduced upon intraperitoneal administration of a chimeric anti-PDGF-CC antibody. In summary, we report on high affinity monoclonal antibodies against PDGF-CC that have therapeutic efficacy in vivo.

MACROD2 Haploinsufficiency Impairs Catalytic Activity of PARP1 and Promotes Chromosome Instability and Growth of Intestinal Tumors.

ADP-ribosylation is an important posttranslational protein modification that regulates diverse biological processes, controlled by dedicated transferases and hydrolases. Here, we show that frequent deletions (∼30%) of the MACROD2 mono-ADP-ribosylhydrolase locus in human colorectal cancer cause impaired PARP1 transferase activity in a gene dosage-dependent manner. MACROD2 haploinsufficiency alters DNA repair and sensitivity to DNA damage and results in chromosome instability. Heterozygous and homozygous depletion of Macrod2 enhances intestinal tumorigenesis in ApcMin/+ mice and the growth of human colorectal cancer xenografts. MACROD2 deletion in sporadic colorectal cancer is associated with the extent of chromosome instability, independent of clinical parameters and other known genetic drivers. We conclude that MACROD2 acts as a haploinsufficient tumor suppressor, with loss of function promoting chromosome instability, thereby driving cancer evolution.Significance: Chromosome instability (CIN) is a hallmark of cancer. We identify MACROD2 deletion as a cause of CIN in human colorectal cancer. MACROD2 loss causes repression of PARP1 activity, impairing DNA repair. MACROD2 haploinsufficiency promotes CIN and intestinal tumor growth. Our results reveal MACROD2 as a major caretaker tumor suppressor gene. Cancer Discov; 8(8); 988-1005. ©2018 AACR.See related commentary by Jin and Burkard, p. 921This article is highlighted in the In This Issue feature, p. 899.

Csk-homologous kinase (Chk) is an efficient inhibitor of Src-family kinases but a poor catalyst of phosphorylation of their C-terminal regulatory tyrosine.

BACKGROUND: C-terminal Src kinase (Csk) and Csk-homologous kinase (Chk) are the major endogenous inhibitors of Src-family kinases (SFKs). They employ two mechanisms to inhibit SFKs. First, they phosphorylate the C-terminal tail tyrosine which stabilizes SFKs in a closed inactive conformation by engaging the SH2 domain in cis. Second, they employ a non-catalytic inhibitory mechanism involving direct binding of Csk and Chk to the active forms of SFKs that is independent of phosphorylation of their C-terminal tail. Csk and Chk are co-expressed in many cell types. Contributions of the two mechanisms towards the inhibitory activity of Csk and Chk are not fully clear. Furthermore, the determinants in Csk and Chk governing their inhibition of SFKs by the non-catalytic inhibitory mechanism are yet to be defined. METHODS: We determined the contributions of the two mechanisms towards the inhibitory activity of Csk and Chk both in vitro and in transduced colorectal cancer cells. Specifically, we assayed the catalytic activities of Csk and Chk in phosphorylating a specific peptide substrate and a recombinant SFK member Src. We employed surface plasmon resonance spectroscopy to measure the kinetic parameters of binding of Csk, Chk and their mutants to a constitutively active mutant of the SFK member Hck. Finally, we determined the effects of expression of recombinant Chk on anchorage-independent growth and SFK catalytic activity in Chk-deficient colorectal cancer cells. RESULTS: Our results revealed Csk as a robust enzyme catalysing phosphorylation of the C-terminal tail tyrosine of SFKs but a weak non-catalytic inhibitor of SFKs. In contrast, Chk is a poor catalyst of SFK tail phosphorylation but binds SFKs with high affinity, enabling it to efficiently inhibit SFKs with the non-catalytic inhibitory mechanism both in vitro and in transduced colorectal cancer cells. Further analyses mapped some of the determinants governing this non-catalytic inhibitory mechanism of Chk to its kinase domain. CONCLUSIONS: SFKs are activated by different upstream signals to adopt multiple active conformations in cells. SFKs adopting these conformations can effectively be constrained by the two complementary inhibitory mechanisms of Csk and Chk. Furthermore, the lack of this non-catalytic inhibitory mechanism accounts for SFK overactivation in the Chk-deficient colorectal cancer cells.

Cross-species analysis of Fc engineered anti-Lewis-Y human IgG1 variants in human neonatal receptor transgenic mice reveal importance of S254 and Y436 in binding human neonatal Fc receptor.

IgG has a long half-life through engagement of its Fc region with the neonatal Fc receptor (FcRn). The FcRn binding site on IgG1 has been shown to contain I253 and H310 in the CH2 domain and H435 in the CH3 domain. Altering the half-life of IgG has been pursued with the aim to prolong or reduce the half-life of therapeutic IgGs. More recent studies have shown that IgGs bind differently to mouse and human FcRn. In this study we characterize a set of hu3S193 IgG1 variants with mutations in the FcRn binding site. A double mutation in the binding site is necessary to abrogate binding to murine FcRn, whereas a single mutation in the FcRn binding site is sufficient to no longer detect binding to human FcRn and create hu3S193 IgG1 variants with a half-life similar to previously studied hu3S193 F(ab')2 (t1/2ß, I253A, 12.23 h; H310A, 12.94; H435A, 12.57; F(ab')2, 12.6 h). Alanine substitutions in S254 in the CH2 domain and Y436 in the CH3 domain showed reduced binding in vitro to human FcRn and reduced elimination half-lives in huFcRn transgenic mice (t1/2ß, S254A, 37.43 h; Y436A, 39.53 h; wild-type, 83.15 h). These variants had minimal effect on half-life in BALB/c nu/nu mice (t1/2ß, S254A, 119.9 h; Y436A, 162.1 h; wild-type, 163.1 h). These results provide insight into the interaction of human Fc by human FcRn, and are important for antibody-based therapeutics with optimal pharmacokinetics for payload strategies used in the clinic.

Synthesis of an inositol hexakisphosphate (IP6) affinity probe to study the interactome from a colon cancer cell line.

Inositol hexakisphosphate (InsP6 or IP6) is an important signalling molecule in vesicular trafficking, neurotransmission, immune responses, regulation of protein kinases and phosphatases, activation of ion channels, antioxidant functions and anticancer activities. An IP6 probe was synthesised from myo-inositol via a derivatised analogue, which was immobilised through a terminal amino group onto Dynabeads. Systematic analysis of the IP6 interactome has been performed using the IP6 affinity probe using cytosolic extracts from the LIM1215 colonic carcinoma cell line. LC/MS/MS analysis identified 77 proteins or protein complexes that bind to IP6 specifically, including AP-2 complex proteins and ß-arrestins as well as a number of novel potential IP6 interacting proteins. Bioinformatic enrichment analysis of the IP6 interactome reinforced the concept that IP6 regulates a number of biological processes including cell cycle and division, signal transduction, intracellular protein transport, vesicle-mediated transport and RNA splicing.

The C-terminal tail inhibitory phosphorylation sites of PTEN regulate its intrinsic catalytic activity and the kinetics of its binding to phosphatidylinositol-4,5-bisphosphate.

Dephosphorylation of four major C-terminal tail sites and occupancy of the phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]-binding site of PTEN cooperate to activate its phospholipid phosphatase activity and facilitate its recruitment to plasma membrane. Our investigation of the mechanism by which phosphorylation of these C-terminal sites controls the PI(4,5)P2-binding affinity and catalytic activity of PTEN resulted in the following findings. First, dephosphorylation of all four sites leads to full activation; and phosphorylation of any one site significantly reduces the intrinsic catalytic activity of PTEN. These findings suggest that coordinated inhibition of the upstream protein kinases and activation of the protein phosphatases targeting the four sites are needed to fully activate PTEN phosphatase activity. Second, PI(4,5)P2 cannot activate the phosphopeptide phosphatase activity of PTEN, suggesting that PI(4,5)P2 can only activate the phospholipid phosphatase activity but not the phosphoprotein phosphatase activity of PTEN. Third, dephosphorylation of all four sites significantly decreases the affinity of PTEN for PI(4,5)P2. Since PI(4,5)P2 is a major phospholipid co-localizing with the phospholipid- and phosphoprotein-substrates in plasma membrane, we hypothesise that the reduced affinity facilitates PTEN to "hop" on the plasma membrane to dephosphorylate these substrates.

Plasmodium falciparum coronin organizes arrays of parallel actin filaments potentially guiding directional motility in invasive malaria parasites.

BACKGROUND: Gliding motility in Plasmodium parasites, the aetiological agents of malaria disease, is mediated by an actomyosin motor anchored in the outer pellicle of the motile cell. Effective motility is dependent on a parasite myosin motor and turnover of dynamic parasite actin filaments. To date, however, the basis for directional motility is not known. Whilst myosin is very likely orientated as a result of its anchorage within the parasite, how actin filaments are orientated to facilitate directional force generation remains unexplained. In addition, recent evidence has questioned the linkage between actin filaments and secreted surface antigens leaving the way by which motor force is transmitted to the extracellular milieu unknown. Malaria parasites possess a markedly reduced repertoire of actin regulators, among which few are predicted to interact with filamentous (F)-actin directly. One of these, PF3D7_1251200, shows strong homology to the coronin family of actin-filament binding proteins, herein referred to as PfCoronin. METHODS: Here the N terminal beta propeller domain of PfCoronin (PfCor-N) was expressed to assess its ability to bind and bundle pre-formed actin filaments by sedimentation assay, total internal reflection fluorescence (TIRF) microscopy and confocal imaging as well as to explore its ability to bind phospholipids. In parallel a tagged PfCoronin line in Plasmodium falciparum was generated to determine the cellular localization of the protein during asexual parasite development and blood-stage merozoite invasion. RESULTS: A combination of biochemical approaches demonstrated that the N-terminal beta-propeller domain of PfCoronin is capable of binding F-actin and facilitating formation of parallel filament bundles. In parasites, PfCoronin is expressed late in the asexual lifecycle and localizes to the pellicle region of invasive merozoites before and during erythrocyte entry. PfCoronin also associates strongly with membranes within the cell, likely mediated by interactions with phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) at the plasma membrane. CONCLUSIONS: These data suggest PfCoronin may fulfil a key role as the critical determinant of actin filament organization in the Plasmodium cell. This raises the possibility that macro-molecular organization of actin mediates directional motility in gliding parasites.

Activation of Src family tyrosine kinases by ferric ions.

The Src-family tyrosine kinases (SFKs) are oncogenic enzymes that contribute to the initiation and progression of many types of cancer. In normal cells, SFKs are kept in an inactive state mainly by phosphorylation of a consensus regulatory tyrosine near the C-terminus (Tyr(530) in the SFK c-Src). As recent data indicate that tyrosine modification enhances binding of metal ions, the hypothesis that SFKs might be regulated by metal ions was investigated. The c-Src C-terminal peptide bound two Fe(3+) ions with affinities at pH4.0 of 33 and 252µM, and phosphorylation increased the affinities at least 10-fold to 1.4 and 23µM, as measured by absorbance spectroscopy. The corresponding phosphorylated peptide from the SFK Lyn bound two Fe(3+) ions with much higher affinities (1.2pM and 160nM) than the Src C-terminal peptide. Furthermore, when Lyn or Hck kinases, which had been stabilised in the inactive state by phosphorylation of the C-terminal regulatory tyrosine, were incubated with Fe(3+) ions, a significant enhancement of kinase activity was observed. In contrast Lyn or Hck kinases in the unphosphorylated active state were significantly inhibited by Fe(3+) ions. These results suggest that Fe(3+) ions can regulate SFK activity by binding to the phosphorylated C-terminal regulatory tyrosine.

A mechanism for actin filament severing by malaria parasite actin depolymerizing factor 1 via a low affinity binding interface.

Actin depolymerizing factor (ADF)/cofilins are essential regulators of actin turnover in eukaryotic cells. These multifunctional proteins facilitate both stabilization and severing of filamentous (F)-actin in a concentration-dependent manner. At high concentrations ADF/cofilins bind stably to F-actin longitudinally between two adjacent actin protomers forming what is called a decorative interaction. Low densities of ADF/cofilins, in contrast, result in the optimal severing of the filament. To date, how these two contrasting modalities are achieved by the same protein remains uncertain. Here, we define the proximate amino acids between the actin filament and the malaria parasite ADF/cofilin, PfADF1 from Plasmodium falciparum. PfADF1 is unique among ADF/cofilins in being able to sever F-actin but do so without stable filament binding. Using chemical cross-linking and mass spectrometry (XL-MS) combined with structure reconstruction we describe a previously overlooked binding interface on the actin filament targeted by PfADF1. This site is distinct from the known binding site that defines decoration. Furthermore, total internal reflection fluorescence (TIRF) microscopy imaging of single actin filaments confirms that this novel low affinity site is required for F-actin severing. Exploring beyond malaria parasites, selective blocking of the decoration site with human cofilin (HsCOF1) using cytochalasin D increases its severing rate. HsCOF1 may therefore also use a decoration-independent site for filament severing. Thus our data suggest that a second, low affinity actin-binding site may be universally used by ADF/cofilins for actin filament severing.

Immunopurification of adenomatous polyposis coli (APC) proteins.

BACKGROUND: The adenomatous polyposis coli (APC) tumour suppressor gene encodes a 2843 residue (310 kDa) protein. APC is a multifunctional protein involved in the regulation of ß-catenin/Wnt signalling, cytoskeletal dynamics and cell adhesion. APC mutations occur in most colorectal cancers and typically result in truncation of the C-terminal half of the protein. RESULTS: In order to investigate the biophysical properties of APC, we have generated a set of monoclonal antibodies which enable purification of recombinant forms of APC. Here we describe the characterisation of these anti-APC monoclonal antibodies (APC-NT) that specifically recognise endogenous APC both in solution and in fixed cells. Full-length APC(1-2843) and cancer-associated, truncated APC proteins, APC(1-1638) and APC(1-1311) were produced in Sf9 insect cells. CONCLUSIONS: Recombinant APC proteins were purified using a two-step affinity approach using our APC-NT antibodies. The purification of APC proteins provides the basis for detailed structure/function analyses of full-length, cancer-truncated and endogenous forms of the protein.

A novel retro-inverso peptide is a preferential JNK substrate-competitive inhibitor.

A novel 18 amino acid peptide PYC98 was demonstrated to inhibit JNK1 activity toward c-Jun. We observed a 5-fold increase in the potency of the retro-inverso form, D-PYC98 (a D-amino acid peptide in the reversed sequence) when compared with the inhibition achieved by L-PYC98, prompting our further evaluation of the D-PYC98 inhibitory mechanism. In vitro assays revealed that, in addition to the inhibition of c-Jun phosphorylation, D-PYC98 inhibited the JNK1-mediated phosphorylation of an EGFR-derived peptide, the ATF2 transcription factor, and the microtubule-regulatory protein DCX. JNK2 and JNK3 activities toward c-Jun were also inhibited, and surface plasmon resonance analysis confirmed the direct interaction of D-PYC98 and JNK1. Further kinetics analyses revealed the non-ATP competitive mechanism of action of D-PYC98 as a JNK1 inhibitor. The targeting of the JNK1 common docking site by D-PYC98 was confirmed by the competition of binding by TIJIP. However, as mutations of JNK1 R127 and E329 within the common docking domain did not impact on the affinity of the interaction with D-PYC98 measured by surface plasmon resonance analysis, other residues in the common docking site appear to contribute to the JNK1 interaction with D-PYC98. Furthermore, we found that D-PYC98 inhibited the related kinase p38 MAPK, suggesting a broader interest in developing D-PYC98 for possible therapeutic applications. Lastly, in evaluating the efficacy of this peptide to act as a substrate competitive inhibitor in cells, we confirmed that the cell-permeable D-PYC98-TAT inhibited c-Jun Ser63 phosphorylation during hyperosmotic stress. Thus, D-PYC98-TAT is a novel cell-permeable JNK inhibitor.

The PI(3)P interactome from a colon cancer cell.

A comprehensive analysis of the phosphoinositide interactome has been performed using an ω-amino analogue of phosphatidylinositol 3-phosphate (PI(3)P immobilised onto Affi-10 beads for use as an affinity absorbent for cytosolic, membrane and nuclear extracts from the LIM1215 colonic carcinoma cell line. Affinity/LC/MS/MS experiments allowed the identification of 681 proteins/protein complexes which interact with PI(3)P. Protein domain enrichment analysis identified proteins possessing PI(3)P (e.g., FYVE, PX, PH), PIP and PIP/phospholipid binding domains along with small GTPases, GTPase regulators, kinases and SH2/SH3 containing proteins. Functional and pathway enrichment analyses highlighted the major role of PI(3)P in endocytosis dynamics and vesicular trafficking, intracellular cell signalling regulation, cell division and cytokinesis. BIOLOGICAL SIGNIFICANCE: This study provides an initial detailed assessment of the phosphatidylinositol 3-phosphate (PI(3)P) interactome, highlights the major role of PI(3)P in endocytosis dynamics and vesicular trafficking, cell intracellular regulation, signalling and cytokinesis and suggests potential PI(3)P specificity for further biochemical and biological characterisation.

mTORC1 inhibition restricts inflammation-associated gastrointestinal tumorigenesis in mice.

Gastrointestinal cancers are frequently associated with chronic inflammation and excessive secretion of IL-6 family cytokines, which promote tumorigenesis through persistent activation of the GP130/JAK/STAT3 pathway. Although tumor progression can be prevented by genetic ablation of Stat3 in mice, this transcription factor remains a challenging therapeutic target with a paucity of clinically approved inhibitors. Here, we uncovered parallel and excessive activation of mTOR complex 1 (mTORC1) alongside STAT3 in human intestinal-type gastric cancers (IGCs). Furthermore, in a preclinical mouse model of IGC, GP130 ligand administration simultaneously activated mTORC1/S6 kinase and STAT3 signaling. We therefore investigated whether mTORC1 activation was required for inflammation-associated gastrointestinal tumorigenesis. Strikingly, the mTORC1-specific inhibitor RAD001 potently suppressed initiation and progression of both murine IGC and colitis-associated colon cancer. The therapeutic effect of RAD001 was associated with reduced tumor vascularization and cell proliferation but occurred independently of STAT3 activity. We analyzed the mechanism of GP130-mediated mTORC1 activation in cells and mice and revealed a requirement for JAK and PI3K activity but not for GP130 tyrosine phosphorylation or STAT3. Our results suggest that GP130-dependent activation of the druggable PI3K/mTORC1 pathway is required for inflammation-associated gastrointestinal tumorigenesis. These findings advocate clinical application of PI3K/mTORC1 inhibitors for the treatment of corresponding human malignancies.

Ligand binding induces a conformational change in epidermal growth factor receptor dimers.

The activation of the epidermal growth factor receptor (EGFR) kinase requires ligand binding to the extracellular domain (ECD). Previous reports demonstrate that the EGFR-ECD can be crystallized in two conformations - a tethered monomer or, in the presence of ligand, an untethered back-to-back dimer. We use Biosensor analysis to demonstrate that even in the monomeric state different C-terminal extensions of both truncated (EGFR(1-501))-ECD and full-length EGFR(1-621)-ECD can change the conformation of the ligand-binding site. The binding of a monoclonal antibody mAb806, which recognizes the dimer interface, to the truncated EGFR(1-501)-Fc fusion protein is reduced in the presence of ligand, consistent with a change in conformation. On the cell surface, the presence of erythroblastosis B2 (erbB2) increases the binding of mAb806 to the EGFR. The conformation of the erbB2: EGFR heterodimer interface changes when the cells are treated with epidermal growth factor (EGF). We propose that ligand induces kinase-inactive, pre-formed EGFR dimers and heterodimers to change conformation leading to kinase-active tetramers, where kinase activation occurs via an asymmetric interaction between EGFR dimers.

Identification of a Wnt-induced protein complex by affinity proteomics using an antibody that recognizes a sub-population of ß-catenin.

ß-catenin is a signaling protein with diverse functions in cell adhesion and Wnt signaling. Although ß-catenin has been shown to participate in many protein-protein interactions, it is not clear which combinations of ß-catenin-interacting proteins form discrete complexes. We have generated a novel antibody, termed 4B3, which recognizes only a small subset of total cellular ß-catenin. Affinity proteomics using 4B3, in combination with subcellular fractionation, has allowed us to define a discrete trimeric complex of ß-catenin, α-catenin and the tumor suppressor APC, which forms in the cytoplasm in response to Wnt signaling. Depletion of the limiting component of this complex, APC, implicates the complex in mediating Wnt-induced changes in cell-cell adhesion. APC is also essential for N-terminal phosphorylation of ß-catenin within this complex. Each component of ß-catenin/APC/α-catenin complex co-exists in other protein complexes, thus use of a selective antibody for affinity proteomics has allowed us to go beyond the generation of a list of potential ß-catenin-interacting proteins, and define when and where a specific complex forms.

Defining the substrate specificity determinants recognized by the active site of C-terminal Src kinase-homologous kinase (CHK) and identification of ß-synuclein as a potential CHK physiological substrate.

C-Terminal Src kinase-homologous kinase (CHK) exerts its tumor suppressor function by phosphorylating the C-terminal regulatory tyrosine of the Src-family kinases (SFKs). The phosphorylation suppresses their activity and oncogenic action. In addition to phosphorylating SFKs, CHK also performs non-SFK-related functions by phosphorylating other cellular protein substrates. To define these non-SFK-related functions of CHK, we used the "kinase substrate tracking and elucidation" method to search for its potential physiological substrates in rat brain cytosol. Our search revealed ß-synuclein as a potential CHK substrate, and Y127 in ß-synuclein as the preferential phosphorylation site. Using peptides derived from ß-synuclein and positional scanning combinatorial peptide library screening, we defined the optimal substrate phosphorylation sequence recognized by the CHK active site to be E-x-[Φ/E/D]-Y-Φ-x-Φ, where Φ and x represent hydrophobic residues and any residue, respectively. Besides ß-synuclein, cellular proteins containing motifs resembling this sequence are potential CHK substrates. Intriguingly, the CHK-optimal substrate phosphorylation sequence bears little resemblance to the C-terminal tail sequence of SFKs, indicating that interactions between the CHK active site and the local determinants near the C-terminal regulatory tyrosine of SFKs play only a minor role in governing specific phosphorylation of SFKs by CHK. Our results imply that recognition of SFKs by CHK is mainly governed by interactions between motifs located distally from the active site of CHK and determinants spatially separate from the C-terminal regulatory tyrosine in SFKs. Thus, besides assisting in the identification of potential CHK physiological substrates, our findings shed new light on how CHK recognizes SFKs and other protein substrates.

Minimal requirements for actin filament disassembly revealed by structural analysis of malaria parasite actin-depolymerizing factor 1.

Malaria parasite cell motility is a process that is dependent on the dynamic turnover of parasite-derived actin filaments. Despite its central role, actin's polymerization state is controlled by a set of identifiable regulators that is markedly reduced compared with those of other eukaryotic cells. In Plasmodium falciparum, the most virulent species that affects humans, this minimal repertoire includes two members of the actin-depolymerizing factor/cofilin (AC) family of proteins, P. falciparum actin-depolymerizing factor 1 (PfADF1) and P. falciparum actin-depolymerizing factor 2. This essential class of actin regulator is involved in the control of filament dynamics at multiple levels, from monomer binding through to filament depolymerization and severing. Previous biochemical analyses have suggested that PfADF1 sequesters monomeric actin but, unlike most eukaryotic counterparts, has limited potential to bind or depolymerize filaments. The molecular basis for these unusual properties and implications for parasite cell motility have not been established. Here we present the crystal structure of an apicomplexan AC protein, PfADF1. We show that PfADF1 lacks critical residues previously implicated as essential for AC-mediated actin filament binding and disassembly, having a substantially reduced filament-binding loop and C-terminal α4 helix. Despite this divergence in structure, we demonstrate that PfADF1 is capable of efficient actin filament severing. Furthermore, this severing occurs despite PfADF1's low binding affinity for filaments. Comparative structural analysis along with biochemical and microscopy evidence establishes that severing is reliant on the availability of an exposed basic residue in the filament-binding loop, a conserved minimal requirement that defines AC-mediated filament disassembly across eukaryotic cells.

PHLDA1 expression marks the putative epithelial stem cells and contributes to intestinal tumorigenesis.

Studies employing mouse models have identified crypt base and position +4 cells as strong candidates for intestinal epithelial stem cells. Equivalent cell populations are thought to exist in the human intestine; however robust and specific protein markers are lacking. Here, we show that in the human small and large intestine, PHLDA1 is expressed in discrete crypt base and some position +4 cells. In small adenomas, PHLDA1 was expressed in a subset of undifferentiated and predominantly Ki-67-negative neoplastic cells, suggesting that a basic hierarchy of differentiation is retained in early tumorigenesis. In large adenomas, carcinomas, and metastases PHLDA1 expression became widespread, with increased expression and nuclear localization at invasive margins. siRNA-mediated suppression of PHLDA1 in colon cancer cells inhibited migration and anchorage-independent growth in vitro and tumor growth in vivo. The integrins ITGA2 and ITGA6 were downregulated in response to PHLDA1 suppression, and accordingly cell adhesion to laminin and collagen was significantly reduced. We conclude that PHLDA1 is a putative epithelial stem cell marker in the human small and large intestine and contributes to migration and proliferation in colon cancer cells.

Characterization of a novel JNK (c-Jun N-terminal kinase) inhibitory peptide.

An improved understanding of the roles of protein kinases in intracellular signalling and disease progression has driven significant advances in protein kinase inhibitor discovery. Peptide inhibitors that target the kinase protein substrate-binding site have continued to attract attention. In the present paper, we describe a novel JNK (c-Jun N-terminal kinase) inhibitory peptide PYC71N, which inhibits JNK activity in vitro towards a range of recombinant protein substrates including the transcription factors c-Jun, ATF2 (activating trancription factor 2) and Elk1, and the microtubule regulatory protein DCX (doublecortin). Analysis of cell culture studies confirmed the actions of a cell-permeable version of PYC71 to inhibit c-Jun phosphorylation during acute hyperosmotic stress. The analysis of the in vitro data for the kinetics of this inhibition indicated a substrate-inhibitor complex-mediated inhibition of JNK by PYC71N. Alanine-scanning replacement studies revealed the importance of two residues (PYC71N Phe9 or Phe11 within an FXF motif) for JNK inhibition. The importance of these residues was confirmed through interaction studies showing that each change decreased interaction of the peptide with c-Jun. Furthermore, PYC71N interacted with both non-phosphorylated (inactive) JNK1 and the substrate c-Jun, but did not recognize active JNK1. In contrast, a previously characterized JNK-inhibitory peptide TIJIP [truncated inhibitory region of JIP (JNK-interacting protein)], showed stronger interaction with active JNK1. Competition binding analysis confirmed that PYC71N inhibited the interaction of c-Jun with JNK1. Taken together, the results of the present study define novel properties of the PYC71N peptide as well as differences from the characterized TIJIP, and highlight the value of these peptides to probe the biochemistry of JNK-mediated substrate interactions and phosphorylation.