Molecular insights into the regulatory landscape of PKC-related kinase-2 (PRK2/PKN2) using targeted small compounds.

The PKC-related kinases (PRKs, also termed PKNs) are important in cell migration, cancer, hepatitis C infection, and nutrient sensing. They belong to a group of protein kinases called AGC kinases that share common features like a C-terminal extension to the catalytic domain comprising a hydrophobic motif. PRKs are regulated by N-terminal domains, a pseudosubstrate sequence, Rho-binding domains, and a C2 domain involved in inhibition and dimerization, while Rho and lipids are activators. We investigated the allosteric regulation of PRK2 and its interaction with its upstream kinase PDK1 using a chemical biology approach. We confirmed the phosphoinositide-dependent protein kinase 1 (PDK1)-interacting fragment (PIF)-mediated docking interaction of PRK2 with PDK1 and showed that this interaction can be modulated allosterically. We showed that the polypeptide PIFtide and a small compound binding to the PIF-pocket of PRK2 were allosteric activators, by displacing the pseudosubstrate PKL region from the active site. In addition, a small compound binding to the PIF-pocket allosterically inhibited the catalytic activity of PRK2. Together, we confirmed the docking interaction and allostery between PRK2 and PDK1 and described an allosteric communication between the PIF-pocket and the active site of PRK2, both modulating the conformation of the ATP-binding site and the pseudosubstrate PKL-binding site. Our study highlights the allosteric modulation of the activity and the conformation of PRK2 in addition to the existence of at least two different complexes between PRK2 and its upstream kinase PDK1. Finally, the study highlights the potential for developing allosteric drugs to modulate PRK2 kinase conformations and catalytic activity.

The role of PKN1 in glioma pathogenesis and the antiglioma effect of raloxifene targeting PKN1.

PKN1 (protein kinase N1), a serine/threonine protein kinase family member, is associated with various cancers. However, the role of PKN1 in gliomas has rarely been studied. We suggest that PKN1 expression in glioma specimens is considerably upregulated and positively correlates with the histopathological grading of gliomas. Knocking down PKN1 expression in glioblastoma (GBM) cells inhibits GBM cell proliferation, invasion and migration and promotes apoptosis. In addition, yes-associated protein (YAP) expression, an essential effector of the Hippo pathway contributing to the oncogenic role of gliomagenesis, was also downregulated. In contrast, PKN1 upregulation enhances the malignant characteristics of GBM cells and simultaneously upregulates YAP expression. Therefore, PKN1 is a promising therapeutic target for gliomas. Raloxifene (Ralo), a commonly used selective oestrogen-receptor modulator to treat osteoporosis in postmenopausal women, was predicted to target PKN1 according to the bioinformatics team from the School of Mathematics, Tianjin Nankai University. We showed that Ralo effectively targets PKN1, inhibits GBM cells proliferation and migration and sensitizes GBM cells to the major chemotherapeutic drug, Temozolomide. Ralo also reverses the effect of PKN1 on YAP activation. Thus, we confirm that PKN1 contributes to the pathogenesis of gliomas and may be a potential target for Ralo adjuvant glioma therapy.

Discovery of new PKN2 inhibitory chemotypes via QSAR-guided selection of docking-based pharmacophores.

Serine/threonine-protein kinase N2 (PKN2) plays an important role in cell cycle progression, cell migration, cell adhesion and transcription activation signaling processes. In cancer, however, it plays important roles in tumor cell migration, invasion and apoptosis. PKN2 inhibitors have been shown to be promising in treating cancer. This prompted us to model this interesting target using our QSAR-guided selection of docking-based pharmacophores approach where numerous pharmacophores are extracted from docked ligand poses and allowed to compete within the context of QSAR. The optimal pharmacophore was sterically-refined, validated by receiver operating characteristic (ROC) curve analysis and used as virtual search query to screen the National Cancer Institute (NCI) database for new promising anti-PKN2 leads of novel chemotypes. Three low micromolar hits were identified with IC50 values ranging between 9.9 and 18.6 µM. Pharmacological assays showed promising cytotoxic properties for active hits in MTT and wound healing assays against MCF-7 and PANC-1 cancer cells.

Upregulation of PKN1 as a Prognosis Biomarker for Endometrial Cancer.

BACKGROUND: Several markers of survival among endometrial cancer (EC) patients have been proposed, namely, the oncoprotein stathmin, RAF kinase inhibitor (RKIP), Cyclin A, GATA-binding protein 3 (GATA3), and growth and differentiation factor-15 (GDF-15). Their elevated expression correlated significantly with a high stage, serous papillary/clear cell subtypes, and aneuploidy. In a previous study, we reported the elevated expression of the serine/threonine protein kinase N1 (PKN1) in cancerous cells. In the present paper, we studied PKN1 expression in EC tissues from a large cohort of patients, to determine whether PKN1 can serve as a marker for the aggressiveness and prognosis of EC, and/or as a marker of survival among EC patients. METHODS: Tissue samples from EC patients were examined retrospectively for tumor type, tumor size, FIGO stage and grade, depth of invasion in the myometrium, and presence of lymph node metastasis. The PKN1 protein expression in EC cells was assessed by immunohistochemistry. PKN1 mRNA levels were analyzed in publicly available databases, using bioinformatic tools. RESULTS: We found that expression of PKN1 at the mRNA and proteins levels tended to increase in high-grade EC samples (P = .0001 and P = .06, respectively). In addition, patients with metastatic disease had higher PKN1 mRNA levels (P = .02). Moreover, patients with high PKN1 expression could be characterized by poorer survival. CONCLUSIONS: We have shown a trend of the higher PKN1 expression levels in EC patients with poor prognosis. Therefore, PKN1 might be considered as a candidate prognostic marker for EC.

Development of dihydropyrrolopyridinone-based PKN2/PRK2 chemical tools to enable drug discovery.

The Protein Kinase N proteins (PKN1, PKN2 and PKN3) are Rho GTPase effectors. They are involved in several biological processes such as cytoskeleton organization, cell mobility, adhesion, and cell cycle. Recently PKNs have been reported as essential for survival in several tumor cell lines, including prostate and breast cancer. Here, we report the development of dihydropyrrolopyridinone-based inhibitors for PKN2 and its closest homologue, PKN1, and their associated structure-activity relationship (SAR). Our studies identified a range of molecules with high potency exemplified by compound 8 with Ki = 8 nM for PKN2 and 14x selectivity over PKN1. Membrane permeability and target engagement for PKN2 were assessed by a NanoBRET cellular assay. Importantly, good selectivity across the wider human kinome and other kinase family members was achieved. These compounds provide strong starting points for lead optimization to PKN1/2 development compounds.

The structure and function of protein kinase C-related kinases (PRKs).

The protein kinase C-related kinase (PRK) family of serine/threonine kinases, PRK1, PRK2 and PRK3, are effectors for the Rho family small G proteins. An array of studies have linked these kinases to multiple signalling pathways and physiological roles, but while PRK1 is relatively well-characterized, the entire PRK family remains understudied. Here, we provide a holistic overview of the structure and function of PRKs and describe the molecular events that govern activation and autoregulation of catalytic activity, including phosphorylation, protein interactions and lipid binding. We begin with a structural description of the regulatory and catalytic domains, which facilitates the understanding of their regulation in molecular detail. We then examine their diverse physiological roles in cytoskeletal reorganization, cell adhesion, chromatin remodelling, androgen receptor signalling, cell cycle regulation, the immune response, glucose metabolism and development, highlighting isoform redundancy but also isoform specificity. Finally, we consider the involvement of PRKs in pathologies, including cancer, heart disease and bacterial infections. The abundance of PRK-driven pathologies suggests that these enzymes will be good therapeutic targets and we briefly report some of the progress to date.

circ_SEPT9, a newly identified circular RNA, promotes oral squamous cell carcinoma progression through miR-1225/PKN2 axis.

Circular RNAs (circRNAs) represent a newly discovered class of endogenous non-coding RNAs which are widely expressed and play important roles in disease progression. However, the function of circRNAs in oral squamous cell carcinoma (OSCC) still remains largely unknown. In this research, we found that circ_SEPT9 was highly expressed in OSCC cell lines and tumour tissues. Results showed that circ_SEPT9 promoted OSCC proliferation and tumour growth. And, circ_SEPT9 also enhanced the migration and invasion of OSCC cells. Mechanically, we found that circ_SEPT9 acted as a sponge for miR-1225 to rescue PKN2 expression in OSCC cells. Inhibition of circ_SEPT9/miR-1225/PKN2 pathway could effectively block the proliferation and metastasis of OSCC cells. Our study provides strong evidence that circ_SEPT9/miR-1225/PKN2 axis is a promising target for OSCC treatment.

The ubiquitin ligase SspH1 from Salmonella uses a modular and dynamic E3 domain to catalyze substrate ubiquitylation.

SspH/IpaH bacterial effector E3 ubiquitin (Ub) ligases, unrelated in sequence or structure to eukaryotic E3s, are utilized by a wide variety of Gram-negative bacteria during pathogenesis. These E3s function in a eukaryotic environment, utilize host cell E2 ubiquitin-conjugating enzymes of the Ube2D family, and target host proteins for ubiquitylation. Despite several crystal structures, details of Ube2D∼Ub binding and the mechanism of ubiquitin transfer are poorly understood. Here, we show that the catalytic E3 ligase domain of SspH1 can be divided into two subdomains: an N-terminal subdomain that harbors the active-site cysteine and a C-terminal subdomain containing the Ube2D∼Ub-binding site. SspH1 mutations designed to restrict subdomain motions show rapid formation of an E3∼Ub intermediate, but impaired Ub transfer to substrate. NMR experiments using paramagnetic spin labels reveal how SspH1 binds Ube2D∼Ub and targets the E2∼Ub active site. Unexpectedly, hydrogen/deuterium exchange MS shows that the E2∼Ub-binding region is dynamic but stabilized in the E3∼Ub intermediate. Our results support a model in which both subunits of an Ube2D∼Ub clamp onto a dynamic region of SspH1, promoting an E3 conformation poised for transthiolation. A conformational change is then required for Ub transfer from E3∼Ub to substrate.

Development of 2-(4-pyridyl)-benzimidazoles as PKN2 chemical tools to probe cancer.

Kinases are signalling proteins which have proven to be successful targets for the treatment of a variety of diseases, predominantly in cancers. However, only a small proportion of kinases (<20%) have been investigated for their therapeutic viability, likely due to the lack of available chemical tools across the kinome. In this work we describe initial efforts in the development of a selective chemical tool for protein kinase N2 (PKN2), a relatively unexplored kinase of interest in several types of cancer. The most successful compound, 5, has a measured IC50 of 0.064 µM against PKN2, with ca. 17-fold selectivity over close homologue, PKN1.

A Screen for PKN3 Substrates Reveals an Activating Phosphorylation of ARHGAP18.

Protein kinase N3 (PKN3) is a serine/threonine kinase implicated in tumor progression of multiple cancer types, however, its substrates and effector proteins still remain largely understudied. In the present work we aimed to identify novel PKN3 substrates in a phosphoproteomic screen using analog sensitive PKN3. Among the identified putative substrates we selected ARHGAP18, a protein from RhoGAP family, for validation of the screen and further study. We confirmed that PKN3 can phosphorylate ARHGAP18 in vitro and we also characterized the interaction of the two proteins, which is mediated via the N-terminal part of ARHGAP18. We present strong evidence that PKN3-ARHGAP18 interaction is increased upon ARHGAP18 phosphorylation and that the phosphorylation of ARHGAP18 by PKN3 enhances its GAP domain activity and contributes to negative regulation of active RhoA. Taken together, we identified new set of potential PKN3 substrates and revealed a new negative feedback regulatory mechanism of Rho signaling mediated by PKN3-induced ARHGAP18 activation.

Phosphorylation Sites in Protein Kinases and Phosphatases Regulated by Formyl Peptide Receptor 2 Signaling.

FPR1, FPR2, and FPR3 are members of Formyl Peptides Receptors (FPRs) family belonging to the GPCR superfamily. FPR2 is a low affinity receptor for formyl peptides and it is considered the most promiscuous member of this family. Intracellular signaling cascades triggered by FPRs include the activation of different protein kinases and phosphatase, as well as tyrosine kinase receptors transactivation. Protein kinases and phosphatases act coordinately and any impairment of their activation or regulation represents one of the most common causes of several human diseases. Several phospho-sites has been identified in protein kinases and phosphatases, whose role may be to expand the repertoire of molecular mechanisms of regulation or may be necessary for fine-tuning of switch properties. We previously performed a phospho-proteomic analysis in FPR2-stimulated cells that revealed, among other things, not yet identified phospho-sites on six protein kinases and one protein phosphatase. Herein, we discuss on the selective phosphorylation of Serine/Threonine-protein kinase N2, Serine/Threonine-protein kinase PRP4 homolog, Serine/Threonine-protein kinase MARK2, Serine/Threonine-protein kinase PAK4, Serine/Threonine-protein kinase 10, Dual specificity mitogen-activated protein kinase kinase 2, and Protein phosphatase 1 regulatory subunit 14A, triggered by FPR2 stimulation. We also describe the putative FPR2-dependent signaling cascades upstream to these specific phospho-sites.

PKC and PKN in heart disease.

The protein kinase C (PKC) and closely related protein kinase N (PKN) families of serine/threonine protein kinases play crucial cellular roles. Both kinases belong to the AGC subfamily of protein kinases that also include the cAMP dependent protein kinase (PKA), protein kinase B (PKB/AKT), protein kinase G (PKG) and the ribosomal protein S6 kinase (S6K). Involvement of PKC family members in heart disease has been well documented over the years, as their activity and levels are mis-regulated in several pathological heart conditions, such as ischemia, diabetic cardiomyopathy, as well as hypertrophic or dilated cardiomyopathy. This review focuses on the regulation of PKCs and PKNs in different pathological heart conditions and on the influences that PKC/PKN activation has on several physiological processes. In addition, we discuss mechanisms by which PKCs and the closely related PKNs are activated and turned-off in hearts, how they regulate cardiac specific downstream targets and pathways, and how their inhibition by small molecules is explored as new therapeutic target to treat cardiomyopathies and heart failure.

PKN2 in colon cancer cells inhibits M2 phenotype polarization of tumor-associated macrophages via regulating DUSP6-Erk1/2 pathway.

BACKGROUND: Protein kinase N2 (PKN2) is a PKC-related serine/threonine-protein kinase. PKN2 is required for tumor cell migration, invasion and apoptosis. However, the functional role of PKN2 in regulating tumor associated macrophages (TAMs) polarization in colon cancer has never been reported. METHODS: PKN2 expression in human colon cancer tissues was examined with immunohistochemistry (IHC). M1/M2 macrophage signatures were evaluated by RT-PCR, IHC and flow cytometry. The effects of PKN2 on tumor growth and TAM polarization were investigated both in vitro and in vivo. PKN2 targeted cytokines/pathway were analyzed by gene expression analysis and further confirmed by PCR, luciferase assay or western blot. Correlations between PKN2 and transcriptional factors for IL4 and IL10 were confirmed by ChIP-qPCR. The catalytic activities of PKN2 and DUSP6 were determined by kinase activity assay. Interactions between PKN2 and DUSP6 were confirmed by Co-IP. RESULTS: The expression of PKN2 in colon cancer cells predicted a favorable prognosis and was associated with low M2 macrophage content in human colon cancer tissues. PKN2 inhibited tumor growth in mice xenograft model and inhibited M2 phenotype polarization both in vitro and in vivo. Mechanistically, PKN2 suppresses the expression of IL4 and IL10 from colon cancer cells by inhibiting Erk1/2 phosphorylation, which is required for phosphorylation and binding of CREB and Elk-1 to the promoters of IL4 and IL10. DUSP6, which is phosphorylated and activated through direct association with PKN2, suppresses Erk1/2 activation. CONCLUSIONS: The expression of PKN2 in colon cancer cells suppresses tumor associated M2 macrophage polarization and tumor growth. Targeting PKN2 signaling pathway may provide a potential therapeutic strategy for colon cancer.

Protein kinase PKN1 represses Wnt/ß-catenin signaling in human melanoma cells.

Advances in phosphoproteomics have made it possible to monitor changes in protein phosphorylation that occur at different steps in signal transduction and have aided the identification of new pathway components. In the present study, we applied this technology to advance our understanding of the responses of melanoma cells to signaling initiated by the secreted ligand WNT3A. We started by comparing the phosphopeptide patterns of cells treated with WNT3A for different periods of time. Next, we integrated these data sets with the results from a siRNA screen that targeted protein kinases. This integration of siRNA screening and proteomics enabled us to identify four kinases that exhibit altered phosphorylation in response to WNT3A and that regulate a luciferase reporter of ß-catenin-responsive transcription (ß-catenin-activated reporter). We focused on one of these kinases, an atypical PKC kinase, protein kinase N1 (PKN1). Reducing the levels of PKN1 with siRNAs significantly enhances activation of ß-catenin-activated reporter and increases apoptosis in melanoma cell lines. Using affinity purification followed by mass spectrometry, we then found that PKN1 is present in a protein complex with a WNT3A receptor, Frizzled 7, as well as with proteins that co-purify with Frizzled 7. These data establish that the protein kinase PKN1 inhibits Wnt/ß-catenin signaling and sensitizes melanoma cells to cell death stimulated by WNT3A.

Protein Kinase C-Related Kinase (PKN/PRK). Potential Key-Role for PKN1 in Protection of Hypoxic Neurons.

Serine/threonine protein kinase C-related kinase (PKN/PRK) is a family of three isoenzymes (PKN1, PKN2, PKN3), which are widely distributed in eukaryotic organisms and share the same overall domain structure. The Nterminal region encompasses a conserved repeated domain, termed HR1a-c as well as a HR2/C2 domain. The serine/threonine kinase domain is found in the C-terminal region of the protein and shows high sequence homology to other members of the PKC superfamily. In neurons, PKN1 is the most abundant isoform and has been implicated in a variety of functions including cytoskeletal organization and neuronal differentiation and its deregulation may contribute to neuropathological processes such as amyotrophic lateral sclerosis and Alzheimer's disease. We have recently identified a candidate role of PKN1 in the regulation of neuroprotective processes during hypoxic stress. Our key findings were that: 1) the activity of PKN1 was significantly increased by hypoxia (1% O2) and neurotrophins (nerve growth factor and purine nucleosides); 2) Neuronal cells, deficient of PKN1 showed a decrease of cell viability and neurite formation along with a disturbance of the F-actinassociated cytoskeleton; 3) Purine nucleoside-mediated neuroprotection during hypoxia was severely hampered in PKN1 deficient neuronal cells, altogether suggesting a potentially critical role of PKN1 in neuroprotective processes. This review gives an up-to-date overview of the PKN family with a special focus on the neuroprotective role of PKN1 in hypoxia.

High-throughput chemogenetic drug screening reveals PKC-RhoA/PKN as a targetable signaling vulnerability in GNAQ-driven uveal melanoma.

Uveal melanoma (UM) is the most prevalent cancer of the eye in adults, driven by activating mutation of GNAQ/GNA11; however, there are limited therapies against UM and metastatic UM (mUM). Here, we perform a high-throughput chemogenetic drug screen in GNAQ-mutant UM contrasted with BRAF-mutant cutaneous melanoma, defining the druggable landscape of these distinct melanoma subtypes. Across all compounds, darovasertib demonstrates the highest preferential activity against UM. Our investigation reveals that darovasertib potently inhibits PKC as well as PKN/PRK, an AGC kinase family that is part of the "dark kinome." We find that downstream of the Gαq-RhoA signaling axis, PKN converges with ROCK to control FAK, a mediator of non-canonical Gαq-driven signaling. Strikingly, darovasertib synergizes with FAK inhibitors to halt UM growth and promote cytotoxic cell death in vitro and in preclinical metastatic mouse models, thus exposing a signaling vulnerability that can be exploited as a multimodal precision therapy against mUM.

PKN1 Kinase: A Key Player in Adipocyte Differentiation and Glucose Metabolism.

Adipocyte dysfunction is the driver of obesity and correlates with insulin resistance and the onset of type 2 diabetes. Protein kinase N1 (PKN1) is a serine/threonine kinase that has been shown to contribute to Glut4 translocation to the membrane and glucose transport. Here, we evaluated the role of PKN1 in glucose metabolism under insulin-resistant conditions in primary visceral adipose tissue (VAT) from 31 patients with obesity and in murine 3T3-L1 adipocytes. In addition, in vitro studies in human VAT samples and mouse adipocytes were conducted to investigate the role of PKN1 in the adipogenic maturation process and glucose homeostasis control. We show that insulin-resistant adipocytes present a decrease in PKN1 activation levels compared to nondiabetic control counterparts. We further show that PKN1 controls the adipogenesis process and glucose metabolism. PKN1-silenced adipocytes present a decrease in both differentiation process and glucose uptake, with a concomitant decrease in the expression levels of adipogenic markers, such as PPARγ, FABP4, adiponectin and CEBPα. Altogether, these results point to PKN1 as a regulator of key signaling pathways involved in adipocyte differentiation and as an emerging player of adipocyte insulin responsiveness. These findings may provide new therapeutic approaches for the management of insulin resistance in type 2 diabetes.

Identification of 4-Anilinoquin(az)oline as a Cell-Active Protein Kinase Novel 3 (PKN3) Inhibitor Chemotype.

Deep annotation of a library of 4-anilinoquin(az)olines led to the identification of 7-iodo-N-(3,4,5-trimethoxyphenyl)quinolin-4-amine 16 as a potent inhibitor (IC50 =14 nM) of Protein Kinase Novel 3 (PKN3) with micromolar activity in cells. Compound 16 is a potential tool compound to study the cell biology of PKN3 and its role in pancreatic and prostate cancer and T-cell acute lymphoblastic leukemia. These 4-anilinoquin(az)olines may also be useful tools to uncover the therapeutic potential of PKN3 inhibition in a broad range of diseases.

Disruption of pancreatic stellate cell myofibroblast phenotype promotes pancreatic tumor invasion.

In pancreatic ductal adenocarcinoma (PDAC), differentiation of pancreatic stellate cells (PSCs) into myofibroblast-like cancer-associated fibroblasts (CAFs) can both promote and suppress tumor progression. Here, we show that the Rho effector protein kinase N2 (PKN2) is critical for PSC myofibroblast differentiation. Loss of PKN2 is associated with reduced PSC proliferation, contractility, and alpha-smooth muscle actin (α-SMA) stress fibers. In spheroid co-cultures with PDAC cells, loss of PKN2 prevents PSC invasion but, counter-intuitively, promotes invasive cancer cell outgrowth. PKN2 deletion induces a myofibroblast to inflammatory CAF switch in the PSC matrisome signature both in vitro and in vivo. Further, deletion of PKN2 in the pancreatic stroma induces more locally invasive, orthotopic pancreatic tumors. Finally, we demonstrate that a PKN2KO matrisome signature predicts poor outcome in pancreatic and other solid human cancers. Our data indicate that suppressing PSC myofibroblast function can limit important stromal tumor-suppressive mechanisms, while promoting a switch to a cancer-supporting CAF phenotype.

Local synthesis of the phosphatidylinositol-3,4-bisphosphate lipid drives focal adhesion turnover.

Focal adhesions are multifunctional organelles that couple cell-matrix adhesion to cytoskeletal force transmission and signaling and to steer cell migration and collective cell behavior. Whereas proteomic changes at focal adhesions are well understood, little is known about signaling lipids in focal adhesion dynamics. Through the characterization of cells from mice with a kinase-inactivating point mutation in the class II PI3K-C2ß, we find that generation of the phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2) membrane lipid promotes focal adhesion disassembly in response to changing environmental conditions. We show that reduced growth factor signaling sensed by protein kinase N, an mTORC2 target and effector of RhoA, synergizes with the adhesion disassembly factor DEPDC1B to induce local synthesis of PtdIns(3,4)P2 by PI3K-C2ß. PtdIns(3,4)P2 then promotes turnover of RhoA-dependent stress fibers by recruiting the PtdIns(3,4)P2-dependent RhoA-GTPase-activating protein ARAP3. Our findings uncover a pathway by which cessation of growth factor signaling facilitates cell-matrix adhesion disassembly via a phosphoinositide lipid switch.