Papers by Jonathan Chernoff
Endocrinology, Jun 1, 2014
p21-activated protein kinase-1 (Pak1) plays a role in insulin secretion and glucagon-like peptide... more p21-activated protein kinase-1 (Pak1) plays a role in insulin secretion and glucagon-like peptide-1 (GLP-1) production. Pak1 Ϫ/Ϫ mice were found to carry a defect in ip pyruvate tolerance test (IPPTT), leading us to speculate whether Pak1 represses hepatic gluconeogenesis. We show here that the defect in IPPTT became more severe in aged Pak1 Ϫ/Ϫ mice. In primary hepatocytes, 2,2Ј-dihydroxy-1,1Ј-dinaphthyldisulfide, a potent inhibitor of group I Paks, reduced basal glucose production (GP), attenuated forskolin-or glucagon-stimulated GP, and attenuated the stimulation of forskolin on the expression of Pck1 and G6pc. In addition, the capacity of primary hepatocytes isolated from Pak1 Ϫ/Ϫ mice in GP at the basal level is significantly lower than that of the control littermates. These in vitro observations imply that the direct effect of Paks in hepatocytes is the stimulation of gluconeogenesis and that the impairment in IPPTT in Pak1 Ϫ/Ϫ mice is due to the lack of Pak1 elsewhere. Consecutive ip injection of forskolin for 2 weeks increased gut proglucagon expression, associated with improved IPPTT in aged Pak1 Ϫ/Ϫ mice and wild-type controls. In addition, administration of the DPP-IV (dipeptidyl peptidase-4) inhibitor sitagliptin for 1 week reversed the defect in IPPTT in aged Pak1 Ϫ/Ϫ mice, associated with increased plasma GLP-1 levels. Our observations indicate a potential role of Pak1 in the gut/pancreas/ liver axis in controlling glucose disposal and affirmed the therapeutic application of GLP-1 and DPP-IV inhibitors in attenuating hepatic gluconeogenesis.
F1000Research, Jun 26, 2017
Protein and lipid kinases are deregulated in most, if not all, cancers and are among the most val... more Protein and lipid kinases are deregulated in most, if not all, cancers and are among the most valuable therapeutic targets in these diseases. Despite the introduction of dozens of effective kinase inhibitors into clinical practice, the development of drug resistance remains a major barrier to treatment because of adaption of cellular signaling pathways to bypass targeted kinases. So that the basal and adaptive responses of kinases in cancer can be better understood, new methods have emerged that allow simultaneous and unbiased measurement of the activation state of a substantial fraction of the entire kinome. Here, we discuss such kinome-profiling methodologies, emphasizing the relative strengths and weaknesses of each approach.
Blood Advances, Oct 10, 2017
Pak2 negatively regulates CD11b high Gr1 high MDSC development in mice via both cellintrinsic and... more Pak2 negatively regulates CD11b high Gr1 high MDSC development in mice via both cellintrinsic and extrinsic mechanisms. • Pak2 disruption activates STAT5 while downregulating the expression of IRF8, a well-described myeloid transcription factor. Myeloid-derived suppressor cells (MDSCs) are CD11b 1 Gr1 1 cells that induce T-cell hyporesponsiveness, thus impairing antitumor immunity. We have previously reported that disruption of Pak2, a member of the p21-activated kinases (Paks), in hematopoietic stem/progenitor cells (HSPCs) induces myeloid lineage skewing and expansion of CD11b high Gr1 high cells in mice. In this study, we confirmed that Pak2-KO CD11b high Gr1 high cells suppressed T-cell proliferation, consistent with an MDSC phenotype. Loss of Pak2 function in HSPCs led to (1) increased hematopoietic progenitor cell sensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling, (2) increased MDSC proliferation, (3) decreased MDSC sensitivity to both intrinsic and Fas-Fas ligand-mediated apoptosis, and (4) promotion of MDSCs by Pak2-deficient CD4 1 T cells that produced more interferon g, tumor necrosis factor a, and GM-CSF. Pak2 disruption activated STAT5 while downregulating the expression of IRF8, a well-described myeloid transcription factor. Together, our data reveal a previously unrecognized role of Pak2 in regulating MDSC development via both cell-intrinsic and extrinsic mechanisms. Our findings have potential translational implications, as the efficacy of targeting Paks in cancer therapeutics may be undermined by tumor escape from immune control and/or acceleration of tumorigenesis through MDSC expansion.
Supplementary Table 2 from p21-Activated Kinase 1 Is Required for Efficient Tumor Formation and Progression in a Ras-Mediated Skin Cancer Model
PDF file - 289K, Immunostaining of Pak1 and proliferative signaling markers in SCC
Supplementary Figure 3 from p21-Activated Kinase 1 Is Required for Efficient Tumor Formation and Progression in a Ras-Mediated Skin Cancer Model
PDF file - 126K, Activity of FRAX-597 against group A and group B Paks
Supplementary Figure 4 from p21-Activated Kinase 1 Is Required for Efficient Tumor Formation and Progression in a Ras-Mediated Skin Cancer Model
PDF file - 162K, Proliferation and apoptosis in drug-treated animals
Supplementary Methods from p21-Activated Kinase 1 Is Required for Efficient Tumor Formation and Progression in a Ras-Mediated Skin Cancer Model
PDF file - 153K
Supplementary Table 1 from p21-Activated Kinase 1 Is Required for Efficient Tumor Formation and Progression in a Ras-Mediated Skin Cancer Model
PDF file - 99K, Immunostaining for Pak1 and Pak2 in mouse skin
Supplementary Figure 5 from p21-Activated Kinase 1 Is Required for Efficient Tumor Formation and Progression in a Ras-Mediated Skin Cancer Model
PDF file - 60K, Schematic presentation of Ras-mediated Pak1 signaling
Supplementary Figure 2 from p21-Activated Kinase 1 Is Required for Efficient Tumor Formation and Progression in a Ras-Mediated Skin Cancer Model
PDF file - 232K, Immunohistochemical analysis of P-Erk, P-Akt, P-S6, and beta-catenin in tumors a... more PDF file - 232K, Immunohistochemical analysis of P-Erk, P-Akt, P-S6, and beta-catenin in tumors arising in K5-tTR::tet-Kras mice
Supplementary Figure Legends 1-5, Table Legends 1-3 from p21-Activated Kinase 1 Is Required for Efficient Tumor Formation and Progression in a Ras-Mediated Skin Cancer Model
PDF file - 131K
Supplementary Table 3 from p21-Activated Kinase 1 Is Required for Efficient Tumor Formation and Progression in a Ras-Mediated Skin Cancer Model
PDF file - 58K, Specificity of Pak inhibitors
Two-hybrid dual bait system to discriminate specificity of protein interactions in small GTPases
Elsevier eBooks, 2001
Publisher Summary This chapter discusses the two-hybrid dual bait system to discriminate specific... more Publisher Summary This chapter discusses the two-hybrid dual bait system to discriminate specificity of protein interactions in Small GTPase. Two-hybrid systems are useful tools that help to identify novel protein interactors for “bait” proteins of interest and to dissect the sequence requirements underlying previously defined protein-protein interactions. Because of the large number of proteins in the Ras superfamily, and because of the sometimes extensive sequence homology among different family members, a particular challenge for the two-hybrid system or any protein interactive technique is to devise a means of readily distinguishing interactions specific for individual members of the Ras group from interactions that do not discriminate among family members. To address such issues of specificity, a second-generation form of the two-hybrid system that simultaneously compares the interaction of two distinct baits with one interactive partner, has been developed. The dual bait system successfully discriminated affinity of interactions among these proteins, and is able to select specific high-affinity interacting pairs from seeded pools of low-affinity interacting proteins, suggesting that these reagents would be useful for more demanding library screening and mutational applications. Two-bait two-hybrid systems promise to provide a rapid and convenient means of developing specific mutations that allow the dissection of unique contributions of individual effectors to the biological output of genes of interest.
Circulation Research, Mar 1, 2019
C ardiovascular disease is a major health challenge and re- mains the leading cause of death worl... more C ardiovascular disease is a major health challenge and re- mains the leading cause of death worldwide. 1 Hearts injured from cardiovascular disease eventually progress to heart failure (HF). Despite numerous causative factors, the overwhelming majority of HF cases are the consequence of massive cardiomyocyte loss. 2 Undoubtedly, the high prevalence and financial burden of HF poses an enormous challenge on us to discover new cardioprotective mechanisms with the hope of developing novel life-saving therapies. Because cardiomyocytes are terminally differentiated, they hold very little potential in replication. Therefore, exquisite regulation of cellular homeostasis and integrity reliant Original
Supplementary Figure 1 from p21-Activated Kinase 1 Is Required for Efficient Tumor Formation and Progression in a Ras-Mediated Skin Cancer Model
PDF file - 219K, Immunostaining for Pak1 and Pak2 in mouse skin
Molecular and Cellular Biology, Dec 1, 2015
p21-activated kinases (Paks) have been shown to regulate cytoskeleton rearrangements, cell prolif... more p21-activated kinases (Paks) have been shown to regulate cytoskeleton rearrangements, cell proliferation, attachment, and migration in a variety of cellular contexts, including endothelial cells. However, the role of endothelial Pak in embryo development has not been reported, and currently, there is no consensus on the endothelial function of individual Pak isoforms, in particular p21-activated kinase 2 (Pak2), the main Pak isoform expressed in endothelial cells. In this work, we employ genetic and molecular studies that show that Pak2, but not Pak1, is a critical mediator of development and maintenance of endothelial cell function. Endothelial depletion of Pak2 leads to early embryo lethality due to flawed blood vessel formation in the embryo body and yolk sac. In adult endothelial cells, Pak2 depletion leads to severe apoptosis and acute angiogenesis defects, and in adult mice, endothelial Pak2 deletion leads to increased vascular permeability. Furthermore, ubiquitous Pak2 deletion is lethal in adult mice. We show that many of these defects are mediated through a newly unveiled Pak2/Bmk1 pathway. Our results demonstrate that endothelial Pak2 is essential during embryogenesis and also for adult blood vessel maintenance, and they also pinpoint the Bmk1/ Erk5 pathway as a critical mediator of endothelial Pak2 signaling. T he p21-activated kinases (Paks) are evolutionarily conserved serine/threonine kinases that act downstream of the Rho family GTPases, Rac1 and Cdc42, to regulate numerous cellular processes. p21-activated kinase 1 (Pak1) to Pak3, the group I Paks, are expressed in numerous tissues with Pak1 being predominantly expressed in brain, muscle, gastrointestinal tract, and thyroid and Pak3 being predominantly expressed in brain (1, 2). Compared to the more restricted expression of Pak1 and Pak3, Pak2 is ubiquitously expressed (3-9). Group I Pak family members share a high degree of homology (3), but they may play distinct roles as observed by the significantly different phenotypes of knockout (KO) animal models. While Pak1 KO and Pak3 KO mice are viable (10, 11), Pak2 KO mice are embryonic lethal at embryonic day 8.5 (E8.5) due to multiple developmental abnormalities, including cardiovascular defects (12). The role of Pak in endothelial cell (EC) signaling has been studied in animal models (5, 13) and cultured cells . Pak signaling is critical in regulating EC attachment, migration, and lumen formation (4-9). Furthermore, Paks have been implicated in maintaining the integrity of the endothelial barrier, but conflicting data implicate various Pak isoforms as both positive and negative regulators of maintaining barrier function . A few in vivo studies have specifically implicated Pak2 in vascular pathways. A study in Danio rerio showed that pak2a, one of two Pak2 orthologs in this organism, plays an important role in cerebral vascular maintenance (5). In this study, chemical mutagenesis of the pak2a gene and pak2a-targeting morpholinos caused cerebral hemorrhage, implicating this Pak family member in vascular barrier maintenance. In mice, germ line deletion of Pak2 is embryonic lethal, and embryos display a phenotype that is consistent with vascular defects (12). While these studies suggest that Pak2 may have an essential role in the vasculature, they do not address the endothelial autonomous function of Pak2, since they relied on global Pak2 deletion in the organism. To study the role of Pak2 in EC signaling in vivo, we generated temporal and tissue-specific conditional Pak2 KO mice. In this article, we present data that suggests that endothelial Pak2 expression is a critical requirement for embryo viability and morphogenesis of embryonic and extraembryonic vasculature. In adult mice, inducible endothelial Pak2 KO causes an increase in vascular leaks in the lung and skin. In vitro studies using primary mouse and human ECs show that Pak2 is required for normal cell proliferation, attachment, migration, and sprouting. Pak2 deletion in ECs is underlined by severe cytoskeletal rearrangements as well as by abnormal signaling through Bmk1/Erk5, also known as mitogen-activated protein kinase 7 (MAPK7), and these defects can be partly suppressed by expression of activated Mek5. Our data show that Pak2 is essential during developmental angiogenesis and also for mature vessel homeostasis and reveal an unexpected link between Pak2 signaling and the Bmk1/Erk5 pathway. Mice. B/6SJL.Tg, C57BL/6J, and Tie2-Cre mice were obtained from The Jackson Laboratory, Bar Harbor, ME. CreER T2 mice were a generous gift
Data from p21-Activated Kinase 1 Is Required for Efficient Tumor Formation and Progression in a Ras-Mediated Skin Cancer Model
The RAS genes are the most commonly mutated oncogenes in human cancer and present a particular th... more The RAS genes are the most commonly mutated oncogenes in human cancer and present a particular therapeutic dilemma, as direct targeting of Ras proteins by small molecules has proved difficult. Signaling pathways downstream of Ras, in particular Raf/Mek/Erk and PI3K/Akt/mTOR, are dominated by lipid and protein kinases that provide attractive alternate targets in Ras-driven tumors. As p21-activated kinase 1 (Pak1) has been shown to regulate both these signaling pathways and is itself upregulated in many human cancers, we assessed the role of Pak1 in Ras-driven skin cancer. In human squamous cell carcinoma (SCC), we found a strong positive correlation between advanced stage and grade and PAK1 expression. Using a mouse model of Kras-driven SCC, we showed that deletion of the mouse Pak1 gene led to markedly decreased tumorigenesis and progression, accompanied by near total loss of Erk and Akt activity. Treatment of KrasG12D mice with either of two distinct small molecule Pak inhibitors (PF3758309 and FRAX597) caused tumor regression and loss of Erk and Akt activity. Tumor regression was also seen in mice treated with a specific Mek inhibitor, but not with an Akt inhibitor. These findings establish Pak1 as a new target in KRAS-driven tumors and suggest a mechanism of action through the Erk, but not the Akt, signaling pathway. Cancer Res; 72(22); 5966–75. ©2012 AACR.
Malignant melanoma is characterized by mutations in a number of driver genes, most notably BRAF a... more Malignant melanoma is characterized by mutations in a number of driver genes, most notably BRAF and NRAS. Recently, genomic analyses revealed that 4-9% of sun-exposed melanoma bear activating mutations in RAC1, which encodes a small GTPase that is known to play key roles in cell proliferation, survival, and migration. The RAC1 protein activates several effector pathways, including Group A p21-activated kinases (PAKs), phosphoinositol-3-kinases (PI3Ks), in particular the beta isoform, and the serum-response factor/myocardin-related transcription factor (SRF/MRTF). Having previously shown that inhibition of Group A PAKs impedes oncogenic signaling from RAC1 P29S , we here extend this analysis to examine the roles of PI3Ks and SRF/MITF in melanocytes and/or in a zebrafish model. We demonstrate that a selective Group A PAK inhibitor (Frax-1036) and certain PI3Ks inhibitors (BKM120, TGX221, GSK2636771) impede the growth of melanoma cells driven by mutant RAC1 but not mutant BRAF, however other PI3K inhibitors, including PI3Kα-selective inhibitors are less effective. Similar results were seen in vivo, using embryonic zebrafish development as a readout, but now including an SRF/MRTF inhibitor (CCG-203971). These results suggest that targeting Group A PAKs and/or SRF/MRTF represent promising approach to suppress RAC1 signaling in malignant melanoma.
p21-Activated Kinases Regulate Directional Migration and Cytoskeletal Organization in Human Neutrophils
Blood, Nov 16, 2012
Abstract 834 Neutrophil chemotaxis is controlled by coordinated processes of directional sensing,... more Abstract 834 Neutrophil chemotaxis is controlled by coordinated processes of directional sensing, polarization and motility. This study was designed to characterize the role of p21-activated kinases (PAKs) during the chemotaxis of human primary neutrophils. PAKs are known as effectors of the Rho GTPases Rac and Cdc42. It has been shown that PAK1 and PAK2 are strongly activated downstream of the f-Met-Leu-Phe (fMLP) receptor via Rac (Huang et al., MCB 1998). PAK1 is known to localize in lamellipodia at the leading edge of human neutrophils (Dharmawardhane et al., JLB 1999) and mediate persistent directional migration via Cdc42 in a neutrophil-like cell line (Li et al., Cell 2003). However, little is known about the specific role of PAK isoforms in spatial/temporal regulation of cytoskeletal dynamics in human neutrophils. Our data show that human neutrophils express PAK1, 2 and 4. Under an fMLP gradient, human neutrophils developed morphological polarity with a distinct leading edge and rear, and migrated up the fMLP gradient at the speed of 7.5 ± 0.56 μm/min. Inhibition of Rac or PI3K impaired directionality but did not significantly affect migration speed of chemotaxing neutrophils (6.3 ± 0.56 μm/min or 6.2 ± 0.85 μm/min, respectively). In contrast, neutrophils treated with the PAK inhibitor, PF3758309 (PF), displayed random migration, less polarization and reduced motility (3.1 ± 0.21 μm/min). These results suggest that PAK regulates neutrophil chemotaxis independently of the Rac-PI3K axis. The presence of PF did not abrogate intracellular Ca2+mobilization in fMLP-driven chemotactic condition. Instead, the decreased migratory ability by PAK inhibition was associated with multiple Ca2+ spikes. Immunofluorescence imaging shows that PAK2 but not PAK1, was phosphorylated and translocated from cytosol to actin-rich leading edge in the proximity to GTP-bound Rac within 3 min of fMLP stimulation. Notably, PF treatment resulted in partial neutrophil spreading and actin/myosin II translocation in the absence of extracellular stimuli, suggesting that basal level of PAK phosphorylation may be required for cytoskeletal integrity of resting neutrophils. Neutrophils pretreated with PF displayed less activation and translocation of PAK2 and Rac. In summary, our data demonstrate for the first time the distinct roles of PAK isoforms in human neutrophil morphological polarity and directional migration and suggest that PAK2 is activated downstream of fMLP receptor through Rho-family small GTPases. Disclosures: No relevant conflicts of interest to declare.
Human Molecular Genetics, Jun 1, 2021
Neurofibromatosis Type II (NF2) is an autosomal dominant cancer predisposition syndrome in which ... more Neurofibromatosis Type II (NF2) is an autosomal dominant cancer predisposition syndrome in which germline haploinsufficiency at the NF2 gene confers a greatly increased propensity for tumor development arising from tissues of neural crest derived origin. NF2 encodes the tumor suppressor, Merlin, and its biochemical function is incompletely understood. One well-established function of Merlin is as a negative regulator of group A serine/threonine p21-activated kinases (PAKs). In these studies we explore the role of PAK1 and its closely related paralog, PAK2, both pharmacologically and genetically, in Merlin-deficient Schwann cells and in a genetically engineered mouse model (GEMM) that develops spontaneous vestibular and spinal schwannomas. We demonstrate that PAK1 and PAK2 are both hyper activated in Merlin-deficient murine schwannomas. In preclinical trials, a pan Group A PAK inhibitor, FRAX-1036, transiently reduced PAK1 and PAK2 phosphorylation in vitro, but had insignificant efficacy in vivo. NVS-PAK1-1, a PAK1 selective inhibitor, had a greater but still minimal effect on our GEMM phenotype. However, genetic ablation of Pak1 but not Pak2 reduced tumor formation in our NF2 GEMM. Moreover, germline genetic deletion of Pak1 was well tolerated, while conditional deletion of Pak2 in Schwann cells resulted in significant morbidity and mortality. These data support the further development of PAK1-specific small molecule inhibitors and the therapeutic targeting of PAK1 in vestibular schwannomas and argue against PAK1 and PAK2 existing as functionally redundant protein isoforms in Schwann cells.
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Papers by Jonathan Chernoff