Search Results (23)

Phosphoinositide-binding pleckstrin homology (PH) domains interact with both phospholipids and proteins, often complicating their use as specific lipid biosensors. In this study, we introduced specific mutations into the phosphatidylinositol 3,4,5-trisphosphate (PIP₃)-specific PH domains of protein kinase B (Akt) and general receptor for phosphoinositides 1 (GRP1) that disrupt protein-mediated interactions while preserving lipid binding, in order to enhance biosensor specificity for PIP₃, and evaluated their impact on plasma membrane (PM) localization and lipid-tracking ability. Using bioluminescence resonance energy transfer (BRET) and confocal microscopy, we assessed the localization of PH domains in HEK293A cells under different conditions. While Akt-PH mutants showed minimal deviations from the wild type, GRP1-PH mutants exhibited significantly reduced PM localization both at baseline and after stimulation with epidermal growth factor (EGF), insulin, or vanadate. We further developed tandem mutant GRP1-PH domain constructs to enhance PM PIP₃ avidity. Additionally, our investigation into the influence of ADP ribosylation factor 6 (Arf6) activity on GRP1-PH-based biosensors revealed that while the wild-type sensors were Arf6- dependent, the mutants operated independently of Arf6 activity level. These optimized GRP1-PH constructs provide a refined biosensor system for accurate and selective detection of dynamic PIP₃ signaling, expanding the toolkit for dissecting phosphoinositide-mediated pathways. Full article

The AKT1 oncogene is related to various cancers due to its critical role in the PIC3CA/AKT1 pathway; however, most of the studies screened the hotspot mutation AKT1 (E17K) with various incidences. Low frequency or lack of AKT1 (E17K) mutation was reported in prostate cancer (PC) patients. This study aims to explore genetic alterations in the AKT1 PH domain by extending the sequencing to include AKT1 gene exons 3 and 4. Genomic DNA was extracted from 84 Formalin-Fixed Paraffin-Embedded samples of PC patients in Jordan, and then subjected to PCR and sequencing for the targeted exons. This study revealed the presence of two novel mutations (N53Y and Q59K) and a high frequency of mutations in exon 4, with a lack of mutations in the E17K hotspot. Nine missense and two synonymous mutations were detected in exon 4 (Phe27Tyr, Phe27Leu, Ala58Thr, Ser56Phe, Arg41Trp, Phe35Leu, Asp32Glu, Phe35Tyr, and Gln43Lys) and (Ser56 and Glu40), respectively. Two synonymous mutations were detected in exon 3 (Leu12 and Ser2). It is concluded that there is a high frequency of AKT1 mutation in PC patients in Jordan with two novel missense mutations in the Pleckstrin homology (PH) domain. E17K hotspot mutation was not detected in any tested samples, which underlined the significant role of mutations in other AKT1 exons in PC development. Full article

Studying primordial events in cancer is pivotal for identifying predictive molecular indicators and for targeted intervention. While the involvement of G-protein-coupled receptors (GPCRs) in cancer is growing, GPCR-based therapies are yet rare. Here, we demonstrate the overexpression of protease-activated receptor 2 (PAR2), a GPCR member in the fallopian tubes (FTs) of high-risk BRCA carriers as compared to null in healthy tissues of FT. FTs, the origin of ovarian cancer, are known to express genes of serous tubal intraepithelial carcinoma (STICs), a precursor lesion of high-grade serous carcinoma (HGSC). PAR2 expression in FTs may serve as an early prediction sensor for ovarian cancer. We show now that knocking down Par2 inhibits ovarian cancer peritoneal dissemination in vivo, pointing to the central role of PAR2. Previously we identified pleckstrin homology (PH) binding domains within PAR1,2&4 as critical sites for cancer-growth. These motifs associate with PH-signal proteins via launching a discrete signaling network in cancer. Subsequently, we selected a compound from a library of backbone cyclic peptides generated toward the PAR PH binding motif, namely the lead compound, Pc(4-4). Pc(4-4) binds to the PAR PH binding domain and blocks the association of PH-signal proteins, such as Akt or Etk/Bmx with PAR2. It attenuates PAR2 oncogenic activity. The potent inhibitory function of Pc(4-4) is demonstrated via inhibition of ovarian cancer peritoneal spread in mice. While the detection of PAR2 may serve as a predictor for ovarian cancer, the novel Pc(4-4) compound may serve as a powerful medicament in STICs and ovarian cancer. This is the first demonstration of the involvement of PAR PH binding motif signaling in ovarian cancer and Pc(4-4) as a potential therapy treatment. Full article

The IRS (insulin receptor substrate) family of scaffold proteins includes insulin receptor substrate-4 (IRS4), which is expressed only in a few cell lines, including human kidney, brain, liver, and thymus and some cell lines. Its N-terminus carries a phosphotyrosine-binding (PTB) domain and a pleckstrin homology domain (PH), which distinguishes it as a member of this family. In this paper, we collected data about the molecular mechanisms that explain the relevance of IRS4 in the development of cancer and identify IRS4 differences that distinguish it from IRS1 and IRS2. Search engines and different databases, such as PubMed, UniProt, ENSEMBL and SCANSITE 4.0, were used. We used the name of the protein that it encodes “(IRS-4 or IRS4)”, or the combination of these terms with the word “(cancer)” or “(human)”, for searches. Terms related to specific tumor pathologies (“breast”, “ovary”, “colon”, “lung”, “lymphoma”, etc.) were also used. Despite the lack of knowledge on IRS4, it has been reported that some cancers and benign tumors are characterized by high levels of IRS-4 expression. Specifically, the role of IRS-4 in different types of digestive tract neoplasms, gynecological tumors, lung cancers, melanomas, hematological tumors, and other less common types of cancers has been shown. IRS4 differs from IRS1 and IRS2 in that can activate several oncogenes that regulate the PI3K/Akt cascade, such as BRK and FER, which are characterized by tyrosine kinase-like activity without regulation via extracellular ligands. In addition, IRS4 can activate the CRKL oncogene, which is an adapter protein that regulates the MAP kinase cascade. Knowledge of the role played by IRS4 in cancers at the molecular level, specifically as a platform for oncogenes, may enable the identification and validation of new therapeutic targets. Full article

GRB2-associated binder 1 (GAB1) is the inaugural member of the GAB/DOS family of pleckstrin homology (PH) domain-containing proteins. Upon receiving various stimuli, GAB1 transitions from the cytoplasm to the membrane where it is phosphorylated by a range of kinases. This event recruits SH2 domain-containing proteins like SHP2, PI3K’s p85 subunit, CRK, and others, thereby activating distinct signaling pathways, including MAPK, PI3K/AKT, and JNK. GAB1-deficient embryos succumb in utero, presenting with developmental abnormalities in the heart, placenta, liver, skin, limb, and diaphragm myocytes. Oncogenic mutations have been identified in the context of cancer. GAB1 expression levels are disrupted in various tumors, and elevated levels in patients often portend a worse prognosis in multiple cancer types. This review focuses on GAB1’s influence on cellular transformation particularly in proliferation, evasion of apoptosis, metastasis, and angiogenesis—each of these processes being a cancer hallmark. GAB1 also modulates the resistance/sensitivity to antitumor therapies, making it a promising target for future anticancer strategies. Full article

Phafins are PH (Pleckstrin Homology) and FYVE (Fab1, YOTB, Vac1, and EEA1) domain-containing proteins. The Phafin protein family is classified into two groups based on their sequence homology and functional similarity: Phafin1 and Phafin2. This protein family is unique because both the PH and FYVE domains bind to phosphatidylinositol 3-phosphate [PtdIns(3)P], a phosphoinositide primarily found in endosomal and lysosomal membranes. Phafin proteins act as PtdIns(3)P effectors in apoptosis, endocytic cargo trafficking, and autophagy. Additionally, Phafin2 is recruited to macropinocytic compartments through coincidence detection of PtdIns(3)P and PtdIns(4)P. Membrane-associated Phafins serve as adaptor proteins that recruit other binding partners. In addition to the phosphoinositide-binding domains, Phafin proteins present a poly aspartic acid motif that regulates membrane binding specificity. In this review, we summarize the involvement of Phafins in several cellular pathways and their potential physiological functions while highlighting the similarities and differences between Phafin1 and Phafin2. Besides, we discuss research perspectives for Phafins. Full article

Arap3, a dual GTPase-activating protein (GAP) for the small GTPases Arf6 and RhoA, plays key roles in regulating a wide range of biological processes, including cancer cell invasion and metastasis. It is known that Arap3 is a PI3K effector that can bind directly to PI(3,4,5)P3, and the PI(3,4,5)P3-mediated plasma membrane recruitment is crucial for its function. However, the molecular mechanism of how the protein recognizes PI(3,4,5)P3 remains unclear. Here, using liposome pull-down and surface plasmon resonance (SPR) analysis, we found that the N-terminal first pleckstrin homology (PH) domain (Arap3-PH1) can interact with PI(3,4,5)P3 and, with lower affinity, with PI(4,5)P2. To understand how Arap3-PH1 and phosphoinositide (PIP) lipids interact, we solved the crystal structure of the Arap3-PH1 in the apo form and complex with diC4-PI(3,4,5)P3. We also characterized the interactions of Arap3-PH1 with diC4-PI(3,4,5)P3 and diC4-PI(4,5)P2 in solution by nuclear magnetic resonance (NMR) spectroscopy. Furthermore, we found overexpression of Arap3 could inhibit breast cancer cell invasion in vitro, and the PIPs-binding ability of the PH1 domain is essential for this function. Full article

Despite its low abundance, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) is a key modulator of membrane-associated signaling events in eukaryotic cells. Temporal and spatial regulation of PI(4,5)P₂ concentration can achieve localized increases in the levels of this lipid, which are crucial for the activation or recruitment of peripheral proteins to the plasma membrane. The recent observation of the dramatic impact of physiological divalent cation concentrations on PI(4,5)P₂ clustering, suggests that protein anchoring to the plasma membrane through PI(4,5)P₂ is likely not defined solely by a simple (monomeric PI(4,5)P₂)/(protein bound PI(4,5)P₂) equilibrium, but instead depends on complex protein interactions with PI(4,5)P₂ clusters. The insertion of PI(4,5)P₂-binding proteins within these clusters can putatively modulate protein–protein interactions in the membrane, but the relevance of such effects is largely unknown. In this work, we characterized the impact of Ca²⁺ on the organization and protein–protein interactions of PI(4,5)P₂-binding proteins. We show that, in giant unilamellar vesicles presenting PI(4,5)P₂, the membrane diffusion properties of pleckstrin homology (PH) domains tagged with a yellow fluorescent protein (YFP) are affected by the presence of Ca²⁺, suggesting direct interactions between the protein and PI(4,5)P₂ clusters. Importantly, PH-YFP is found to dimerize in the membrane in the absence of Ca²⁺. This oligomerization is inhibited in the presence of physiological concentrations of the divalent cation. These results confirm that cation-dependent PI(4,5)P₂ clustering promotes interactions between PI(4,5)P₂-binding proteins and has the potential to dramatically influence the organization and downstream interactions of PI(4,5)P₂-binding proteins in the plasma membrane. Full article

The lack of therapeutic alternatives for the treatment of Chagas disease, a neglected disease, drives the discovery of new drugs with trypanocidal activity. Consequently, we conducted in vitro studies using UBMC-4, a potential Trypanosoma cruzi AKT-like pleckstrin homology (PH) domain inhibitory compound found using bioinformatics tools. The half effective concentration (EC₅₀) on intracellular amastigotes was determined at 1.85 ± 1 μM showing low cytotoxicity (LC₅₀) > 40 μM on human cell lines tested. In order to study the lethal effect caused by the compound on epimastigotes, morphological changes were assessed by scanning and transmission electron microscopy. Progressive alterations such as flagellum inactivation, cell size reduction, nuclear structure alteration, condensation of chromatin towards the nuclear periphery, vacuole formation, and mitochondrial swelling with kinetoplast integrity loss were evidenced. In addition, apoptosis-like markers in T. cruzi were assessed by flow cytometry, demonstrating that the effect of UBMC-4 on T. cruzi AKT-like kinase reduced the tolerance to nutritional stress-triggered, apoptosis-like events, including DNA fragmentation, mitochondrial damage, and loss of plasma membrane integrity. After this, UBMC-4 was formulated for oral administration and pharmacokinetics were analyzed in a mouse model. Finally, upon oral administration of 200 mg/kg in mice, we found that a UBMC-4 plasma concentration remaining in circulation beyond 24 h after administration is well described by the two-compartment model. We conclude that UBMC-4 has an effective trypanocidal activity in vitro at low concentrations and this effect is evident in T. cruzi cell structures. In mice, UBMC-4 was well absorbed and reached plasma concentrations higher than the EC₅₀, showing features that would aid in developing a new drug to treat Chagas disease. Full article

The Connector Enhancer of Kinase Suppressor of Ras-2 (CNKSR2), also known as CNK2 or MAGUIN, is a scaffolding molecule that contains functional protein binding domains: Sterile Alpha Motif (SAM) domain, Conserved Region in CNK (CRIC) domain, PSD-95/Dlg-A/ZO-1 (PDZ) domain, Pleckstrin Homology (PH) domain, and C-terminal PDZ binding motif. CNKSR2 interacts with different molecules, including RAF1, ARHGAP39, and CYTH2, and regulates the Mitogen-Activated Protein Kinase (MAPK) cascade and small GTPase signaling. CNKSR2 has been reported to control the development of dendrite and dendritic spines in primary neurons. CNKSR2 is encoded by the CNKSR2 gene located in the X chromosome. CNKSR2 is now considered as a causative gene of the Houge type of X-linked syndromic mental retardation (MRXHG), an X-linked Intellectual Disability (XLID) that exhibits delayed development, intellectual disability, early-onset seizures, language delay, attention deficit, and hyperactivity. In this review, we summarized molecular features, neuronal function, and neurodevelopmental disorder-related variations of CNKSR2. Full article

Bacteriocins synthesis is initiated from an inactive precursor, which is composed of an N-terminal leader peptide attached to a C-terminal pro-peptide. However, leaderless bacteriocins (LLB) do not possess this N-terminal leader peptide nor undergo post-translational modifications. These atypical bacteriocins are observed to be immediately active after their translation in the cytoplasm. However, although considered to be simple, the biosynthetic pathway of LLB remains to be fully understood. Enterocin DD14 (EntDD14) is a two-peptide LLB produced by Enterococcus faecalis 14, which is a strain isolated from meconium. In silico analysis of DNA encoding EntDD14 located a cluster of 10 genes ddABCDEFGHIJ, where ddE and ddF encode the peculiar DdE and DdF proteins, carrying pleckstrin homology (PH) domains. These modules are quite common in Eucarya proteins and are known to be involved in intracellular signaling or cytoskeleton organization. To elucidate their role within the EntDD14 genetic determinants, we constructed deletion mutants of the ddE and ddF genes. As a result, the mutants were unable to export EntDD14 outside of the cytoplasm even though there was a clear expression of structural genes ddAB encoding EntDD14, and genes ddHIJ encoding an ABC transporter. Importantly, in these mutant strains (ΔddE and ΔddF), EntDD14 was detected by mass spectrometry in the intracellular soluble fraction exerting, upon its accumulation, a toxic effect on the producing strain as revealed by cell-counting and confocal microscopy analysis. Taken together, these results clearly indicate that PH domain-containing proteins, such as DdE and DdF, are involved in the transport of the leaderless two-peptide EntDD14. Full article

VPS13 proteins are evolutionarily conserved. Mutations in the four human genes (VPS13A-D) encoding VPS13A-D proteins are linked to developmental or neurodegenerative diseases. The relationship between the specific localization of individual VPS13 proteins, their molecular functions, and the pathology of these diseases is unknown. Here we used a yeast model to establish the determinants of Vps13′s interaction with the membranes of Golgi apparatus. We analyzed the different phenotypes of the arf1-3 arf2Δ vps13∆ strain, with reduced activity of the Arf1 GTPase, the master regulator of Golgi function and entirely devoid of Vps13. Our analysis led us to propose that Vps13 and Arf1 proteins cooperate at the Golgi apparatus. We showed that Vps13 binds to the Arf1 GTPase through its C-terminal Pleckstrin homology (PH)-like domain. This domain also interacts with phosphoinositol 4,5-bisphosphate as it was bound to liposomes enriched with this lipid. The homologous domain of VPS13A exhibited the same behavior. Furthermore, a fusion of the PH-like domain of Vps13 to green fluorescent protein was localized to Golgi structures in an Arf1-dependent manner. These results suggest that the PH-like domains and Arf1 are determinants of the localization of VPS13 proteins to the Golgi apparatus in yeast and humans. Full article

Transmembrane prostate androgen-induced protein (TMEPAI), also known as PMEPA1, is highly expressed in many types of cancer and promotes oncogenic abilities. However, the mechanisms whereby TMEPAI facilitates tumorigenesis are not fully understood. We previously established TMEPAI-knockout (KO) cells from human triple-negative breast cancer (TNBC) cell lines and found that TMEPAI-KO cells showed reduced tumorigenic abilities. Here, we report that TMEPAI-KO cells upregulated the expression of pleckstrin homology (PH) domain and leucine-rich repeat protein phosphatase 1 (PHLPP1) and suppressed AKT Ser473 phosphorylation, which was consistent with TCGA dataset analysis. Additionally, the knockdown (KD) of PHLPP1 in TMEPAI-KO cells partially but significantly rescued AKT Ser473 phosphorylation, as well as in vitro and in vivo tumorigenic activities, thus showing that TMEPAI functions as an oncogenic protein through the regulation of PHLPP1 subsequent to AKT activation. Furthermore, we demonstrated that TMEPAI PPxY (PY) motifs are essential for binding to NEDD4-2, an E3 ubiquitin ligase, and PHLPP1-downregulatory ability. Moreover, TMEPAI enhanced the complex formation of PHLPP1 with NEDD4-2 and PHLPP1 polyubiquitination, which leads to its proteasomal degradation. These findings indicate that the PY motifs of TMEPAI suppress the amount of PHLPP1 and maintain AKT Ser473 phosphorylation at high levels to enhance the tumorigenic potentiality of TNBC. Full article

G protein-coupled receptor kinase 2 (GRK2) is emerging as a key integrative signaling node in a variety of biological processes ranging from cell growth and proliferation to migration and chemotaxis. As such, GRK2 is now implicated as playing a role in the molecular pathogenesis of a broad group of diseases including heart failure, cancer, depression, neurodegenerative disease, and others. In addition to its long-known canonical role in the phosphorylation and desensitization of G protein-coupled receptors (GPCRs), recent studies have shown that GRK2 also modulates a diverse array of other molecular processes via newly identified GRK2 kinase substrates and via a growing number of protein-protein interaction binding partners. GRK2 belongs to the 7-member GRK family. It is a multidomain protein containing a specific N-terminal region (referred to as αN), followed by a regulator of G protein signaling homology (RH) domain, an AGC (Protein kinase A, G, C serine/threonine kinase family) kinase domain, and a C-terminal pleckstrin homology (PH) domain. GPCRs mediate the activity of many regulators of the immune system such as chemokines and leukotrienes, and thus GRK proteins may play key roles in modulating the lymphocyte response to these factors. As one of the predominant GRK family members expressed in immune cells, GRK2′s canonical and noncanonical actions play an especially significant role in normal immune cell function as well as in the development and progression of disorders of the immune system. This review summarizes our current state of knowledge of the roles of GRK2 in lymphocytes. We highlight the diverse functions of GRK2 and discuss how ongoing investigation of GRK2 in lymphocytes may inform the development of new therapies for diseases associated with lymphocyte dysregulation. Full article

MINERVA (melanoma invasion by ERK), also known as FAM129B, is a member of the FAM129 protein family, which is only present in vertebrates. MINERVA is involved in key signaling pathways regulating cell survival, proliferation and apoptosis and found upregulated in many types of cancer promoting invasion. However, the exact function of the protein remains elusive. X-ray crystallographic methods were implemented to determine the crystal structure of MINERVA^ΔC, lacking C-terminal flexible region. Trypsin digestion was required before crystallization to obtain diffraction-quality crystals. While the N-terminal pleckstrin homology (PH) domain exhibits the typical fold of PH domains, lipid binding assay indicates specific affinity towards phosphatidic acid and inositol 3-phosphate. A helix-rich domain that constitutes the rest of the molecule demonstrates a novel L-shaped fold that encompasses the PH domain. The overall structure of MINERVA^ΔC with binding assays and cell-based experiments suggest plasma membrane association of MINERVA and its function seem to be tightly regulated by various motifs within the C-terminal flexible region. Elucidation of MINERVA^ΔC structure presents a novel fold for an α-helix bundle domain that would provide a binding platform for interacting partners. Full article