Biomedical subjects

Jonathan Chernoff

Publications and source records attributed to Jonathan Chernoff.

36 records · Page 2Linked to original sources

PubMed2003

AND-34/BCAR3, a GDP exchange factor whose overexpression confers antiestrogen resistance, activates Rac, PAK1, and the cyclin D1 promoter.

AND-34 is a murine protein that binds by a cdc25-like GDP exchange factor domain to the focal adhesion docking protein p130Cas. Overexpression of either of the human homologues of AND-34 and p130Cas, BCAR3 and BCAR1, respectively, has been reported to induce resistance to antiestrogens in breast cancer cell lines. Here we show that overexpression of AND-34 leads to activation of the Rho family GTPases Cdc42 and Rac. Consistent with these findings, BCAR3 overexpression induced alterations in F-actin distribution and augmented both autophosphorylation and kinase activity of the Cdc42/Rac-responsive serine/threonine kinase PAK1. p130Cas-associated BCAR3 protein was detected in the estrogen-independent breast cancer cell line 578-T, but not in estrogen-dependent MCF7 or ZR-75-1 cells. Stable ZR-75-1 transfectants overexpressing BCAR3, but not vector-only transfectants, grew in the presence of the pure antiestrogen ICI 182,780. Stable transfection with RacV12, a constitutively active form of Rac1, also induced antiestrogen resistance in ZR-75-1 cells. Transient transfection of BCAR3 in estrogen-dependent MCF7 cells induced activation of luciferase constructs containing the proximal 1745 or 163 bp but not 66 bp of the cyclin D1 promoter. Such cyclin D1 promoter activation was inhibited by dominant negative forms of Rac1 and PAK1. Overexpression of the PAK1 autoinhibitory domain (residues 83-149) but not an inactive PAK1 autoinhibitory domain point mutant (L107F) also blocked BCAR3-mediated cyclin D1 activation. These studies suggest that AND-34/BCAR3 induces antiestrogen resistance in breast cancer cell lines by a Rac1- and PAK1-dependent pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Pak1 and PIX regulate contact inhibition during epithelial wound healing.

Wound healing in epithelia requires coordinated cell migration and proliferation regulated by signaling mechanisms that are poorly understood. Here we show that epithelial cells expressing constitutively active or kinase-dead mutants of the Rac/Cdc42 effector Pak1 fail to undergo growth arrest upon wound closure. Strikingly, this phenotype is only observed when the Pak1 kinase mutants are expressed in cells possessing a free lateral surface, i.e. one that is not engaged in contact with neighboring cells. The Pak1 kinase mutants perturb contact inhibition by a mechanism that depends on the Pak-interacting Rac-GEF PIX. In control cells, endogenous activated Pak and PIX translocate from focal complexes to cell-cell contacts during wound closure. This process is abrogated in cells expressing Pak1 kinase mutants. In contrast, Pak1 mutants rendered defective in PIX binding do not impede translocation of activated Pak and PIX, and exhibit normal wound healing. Thus, recruitment of activated Pak and PIX to cell-cell contacts is pivotal to transduction of growth-inhibitory signals from neighboring cells in epithelial wound healing.

Animals↗

PubMed2003

Protein-tyrosine phosphatase 1B mediates the effects of insulin on the actin cytoskeleton in immortalized fibroblasts.

Insulin regulates diverse cellular responses including actin reorganization. The mechanism by which insulin induces formation of lamellipodia in cultured cells is not known but is likely to involve activation of Src family protein-tyrosine kinases. Here we show that protein-tyrosine phosphatase 1B (PTPIB) activates Src, thereby initiating the activation of a Rac-dependent pathway leading to cytoskeletal remodeling. Conversely, expression of a proline to alanine (P309,310A) PTP1B mutant, which cannot activate Src, fails to activate Rho GTPases or cause changes in actin organization. Rat fibroblasts lacking PTP1B expression do not activate Src or Rac in response to insulin and cannot reorganize actin. These results show that PTP1B, best known as a negative regulator of the metabolic effects of insulin, is required for the effects of insulin on actin organization in immortalized fibroblasts.

Actins↗

PubMed2003

Apoptotic phosphorylation of histone H2B is mediated by mammalian sterile twenty kinase.

DNA in eukaryotic cells is associated with histone proteins; hence, hallmark properties of apoptosis, such as chromatin condensation, may be regulated by posttranslational histone modifications. Here we report that phosphorylation of histone H2B at serine 14 (S14) correlates with cells undergoing programmed cell death in vertebrates. We identify a 34 kDa apoptosis-induced H2B kinase as caspase-cleaved Mst1 (mammalian sterile twenty) kinase. Mst1 can phosphorylate H2B at S14 in vitro and in vivo, and the onset of H2B S14 phosphorylation is dependent upon cleavage of Mst1 by caspase-3. These data reveal a histone modification that is uniquely associated with apoptotic chromatin in species ranging from frogs to humans and provide insights into a previously unrecognized physiological substrate for Mst1 kinase. Our data provide evidence for a potential apoptotic "histone code."

Apoptosis↗

PubMed2003

p38 Mitogen-activated protein kinase mediates cell death and p21-activated kinase mediates cell survival during chemotherapeutic drug-induced mitotic arrest.

Activation of the mitotic checkpoint by chemotherapeutic drugs such as taxol causes mammalian cells to arrest in mitosis and then undergo apoptosis. However, the biochemical basis of chemotherapeutic drug-induced cell death is unclear. Herein, we provide new evidence that both cell survival and cell death-signaling pathways are concomitantly activated during mitotic arrest by microtubule-interfering drugs. Treatment of HeLa cells with chemotherapeutic drugs activated both p38 mitogen-activated protein kinase (MAPK) and p21-activated kinase (PAK). p38 MAPK was necessary for chemotherapeutic drug-induced cell death because the p38 MAPK inhibitors SB203580 or SB202190 suppressed cell death. Dominant-active MKK6, a direct activator of p38 MAPK, also induced cell death by stimulating translocation of Bax from the cytosol to the mitochondria in a p38 MAPK-dependent manner. Dominant active PAK suppressed this MKK6-induced cell death. PAK seems to mediate cell survival by phosphorylating Bad, and inhibition of PAK in mitotically arrested cells reduced Bad phosphorylation and increased apoptosis. Our results suggest that therapeutic strategies that suppress PAK-mediated survival signals may improve the efficacy of current cancer chemotherapies by enhancing p38 MAPK-mediated cell death.

Antineoplastic Agents↗

PubMed2003

Rapid induction of dendritic spine morphogenesis by trans-synaptic ephrinB-EphB receptor activation of the Rho-GEF kalirin.

The morphogenesis of dendritic spines, the major sites of excitatory synaptic transmission in the brain, is important in synaptic development and plasticity. We have identified an ephrinB-EphB receptor trans-synaptic signaling pathway which regulates the morphogenesis and maturation of dendritic spines in hippocampal neurons. Activation of the EphB receptor induces translocation of the Rho-GEF kalirin to synapses and activation of Rac1 and its effector PAK. Overexpression of dominant-negative EphB receptor, catalytically inactive kalirin, or dominant-negative Rac1, or inhibition of PAK eliminates ephrin-induced spine development. This novel signal transduction pathway may be critical for the regulation of the actin cytoskeleton controlling spine morphogenesis during development and plasticity.

Blotting, Western↗

PubMed2003

NF2: the wizardry of merlin.

Neurofibromatosis type II (NF2) is an autosomal dominant cancer syndrome characterized by the formation of tumors of the nervous system, particularly schwannomas and meningiomas. The NF2 gene is also implicated in the development of sporadic schwannomas and meningiomas, as well as tumor types seemingly unrelated to the NF2 disorder, such as malignant mesotheliomas. Inactivation of NF2 occurs by a "two-hit" mechanism, as proposed by Al Knudson, and the NF2 gene behaves as a classical tumor suppressor gene. The NF2 gene product, merlin, exhibits homology with the ezrin-radixin-moesin family of membrane-cytoskeleton-linking proteins. During the past several years, there has been intensive investigation aimed at elucidating the mechanisms underlying merlin's functions. In this review, we summarize the involvement of NF2 inactivation in tumorigenesis. We also discuss observations implicating merlin in cell motility and cell proliferation, with a focus on recent findings linking merlin to Rac signaling.

Animals↗

PubMed2003

p21-Activated kinase 5 (Pak5) localizes to mitochondria and inhibits apoptosis by phosphorylating BAD.

Pak5 is the most recently identified and least understood member of the p21-activated kinase (Pak) family. This kinase is known to promote neurite outgrowth in vitro, but its localization, substrates, and effects on cell survival have not been reported. We show here that Pak5 has unique properties that distinguish it from all other members of the Pak family. First, Pak5, unlike Pak1, cannot complement an STE20 mutation in Saccharomyces cerevisiae. Second, Pak5 binds to the GTPases Cdc42 and Rac, but these GTPases do not regulate Pak5 kinase activity, which is constitutive and stronger than any other Pak. Third, Pak5 prevents apoptosis induced by camptothecin and C2-ceramide by phosphorylating BAD on Ser-112 in a protein kinase A-independent manner and prevents the localization of BAD to mitochondria, thereby inhibiting the apoptotic cascade that leads to apoptosis. Finally, we show that Pak5 itself is constitutively localized to mitochondria, and that this localization is independent of kinase activity or Cdc42 binding. These features make Pak5 unique among the Pak family and suggest that it plays an important role in apoptosis through BAD phosphorylation.

Animals↗

PubMed2003

Protein-tyrosine phosphatase 1B as a potential drug target for obesity.

Obesity is increasing at an alarming rate and is considered by the World Health Organization as one of the top 10 epidemics worldwide. Resistance to leptin and insulin are likely to play a central role in obesity; thus, blocking inhibitors of these signaling pathways could prove useful in treating this disorder. Several lines of evidence have converged on protein tyrosine-phosphatase 1B (PTP1B) as one of the most important negative regulators of leptin as well as insulin signaling. Therefore, PTP1B appears to be a promising therapeutic candidate for the treatment of obesity. In this review, we discuss the role of PTP1B in leptin and insulin signaling, as well as its potential as a drug target in the treatment of obesity.

Animals↗

PubMed2002

Tuberin, the tuberous sclerosis complex 2 tumor suppressor gene product, regulates Rho activation, cell adhesion and migration.

Tuberous sclerosis complex (TSC) is a tumor suppressor gene syndrome characterized by seizures, mental retardation, autism, and tumors of the brain, kidney, heart, retina, and skin. TSC is caused by mutations in either TSC1 or TSC2, both of which are tumor suppressor genes. Hamartin, the protein product of TSC1, was found to interact with the ezrin-radixin-moesin family of cytoskeletal proteins and to activate the small GTPase Rho. To determine whether tuberin, the TSC2 product, can also activate Rho, we stably expressed full-length human tuberin in two cell types: MDCK cells and ELT3 cells. ELT3 cells lack endogenous tuberin expression. We found that expression of human tuberin in both MDCK and ELT3 cells was associated with an increase in the amount of Rho-GTP, but not in Rac1-GTP or cdc42-GTP. Tuberin expression increased cell adhesion in both cell types, and decreased chemotactic cell migration in ELT3 cells. In MDCK cells, there was a decrease in the amount of total Focal Adhesion Kinase (FAK) and an increase in the fraction of phosphorylated FAK. These findings demonstrate for the first time that tuberin activates Rho and regulates cell adhesion and migration. Pathways involving Rho activation may have relevance to the clinical manifestations of TSC, including pulmonary lymphangioleiomyomatosis.

Animals↗

PubMed2002

Cell cycle-regulated phosphorylation of p21-activated kinase 1.

Mammalian p21-activated kinase 1 (Pak1) is a highly conserved effector for the small GTPases Cdc42 and Rac1. In lower eukaryotes, Pak1 homologs are regulated during the cell cycle by phosphorylation. Here, we show that Pak1 is phosphorylated during mitosis in mammalian fibroblasts. This phosphorylation occurs at a single site, Thr 212, within a domain that is unique to Pak1. Cdc2 phosphorylates Pak1 at the identical site in vitro, and inhibition of Cdc2 abolishes Pak1 mitotic phosphorylation in vivo, indicating that Cdc2 is the kinase responsible for phosphorylating Pak1 in mitotic cells. Expression of a Pak1 mutant in which Thr 212 is replaced with a phosphomimic (aspartic acid) has marked effects on the rate and extent of postmitotic spreading of fibroblasts. The mitotic phosphorylation of Pak1 does not alter the basal or Rac-stimulated activity of this kinase, but it does affect the coimmunoprecipitation of at least three proteins with Pak1. These findings are the first to implicate a mammalian Pak in cell cycle regulation and suggest that Pakl, as a result of phosphorylation by Cdc2, alters its association with binding partners and/or substrates that are relevant to the morphologic changes associated with cell division.

Animals↗

PubMed2002

p21-activated kinase 1 interacts with and phosphorylates histone H3 in breast cancer cells.

Stimulation of p21-activated kinase-1 (Pak1) signaling promotes motility, invasiveness, anchorage-independent growth and abnormal mitotic assembly in human breast cancer cells. Here, we provide new evidence that, before the onset of mitosis, activated Pak1 is specifically localized with the chromosomes during prophase and on the centrosomes in metaphase and moves to the contraction ring during cytokinesis. To identify mitosis-specific substrates of Pak1, we screened a synchronized G2-M expression library by using a glutathione transferase Pak1 solid-phase-based kinase reaction. This analysis identified histone H3 as a substrate of Pak1 both in vitro and in vivo, and it specifically interacted with Pak1 but not Pak2 or Pak3. Site-directed mutagenesis indicated that Pak1 phosphorylates histone H3 on Ser10. Expressions of the wild-type, or catalytically active, Pak1 caused it to appear at the poles corresponding to mitotic centrosomes in a variety of mammalian cells. Together, these results suggest for the first time that Pak1 interacts with and phosphorylates histone H3 and may thus influence the Pak1-histone H3 pathway, which in turn may influence mitotic events in breast cancer cells.

Breast Neoplasms↗

PubMed2002

Interaction of protein tyrosine phosphatase (PTP) 1B with its substrates is influenced by two distinct binding domains.

We have shown previously that protein tyrosine phosphatase (PTP) 1B interacts with insulin receptor and negatively regulates insulin signalling by an N-terminal binding domain [Dadke, Kusari and Chernoff (2000) J. Biol. Chem. 275, 23642-23647] and it also negatively regulates integrin signalling through a proline-rich region present in the C-terminus [Liu, Hill and Chernoff (1996) J. Biol. Chem. 271, 31290-31295; Liu, Sells and Chernoff (1998) Curr. Biol. 8, 173-176]. Here we show that PTP1B mutants that are defective in Src homology 3 domain binding fully retain the ability to inhibit insulin signalling, whereas mutants defective in insulin-receptor binding fully retain the ability to inhibit integrin signalling. In contrast, both the C-terminal proline-rich region and the tandem tyrosine residues present in the N-terminal region are required for the activation of Src family kinases. These data show that PTP1B can independently regulate insulin and integrin signals, and that Src might represent a convergence point for regulating signal transduction by this phosphatase.

Animals↗

PubMed2002

The evolutionary history of effectors downstream of Cdc42 and Rac.

Cdc42 and Rac--ancient, highly conserved, small GTPases--mediate extracellular signals, triggering changes in transcription and in the actin cytoskeleton. Although dozens of proteins act downstream of these GTPases, a comparison of effector proteins from evolutionarily diverse organisms suggests that six groups of proteins serve as the core machinery for signaling from Cdc42 and Rac.

Animals↗

PubMed2002

High midsummer for small GTPases.

The recent FASEB meeting on 'Small GTPases and Cell dynamics' (Snowmass, 6-11 July, 2002) focused on the emerging connections between small GTPases and the actin and tubulin cytoskeletons, advances in understanding where and when these proteins are activated, and their role in directing intracellular traffic and cell polarity in systems ranging from fibroblasts in Petri dishes to neurons in living organisms. The variety and vigor of the presentations showed that this field of cell biology remains a fertile ground for discovery.

Animals↗

PubMed2002

p21-activated kinases: three more join the Pak.

The p21-activated kinases (Paks) are serine/threonine protein kinases that bind to and, in some cases, are stimulated by activated forms of the small GTPases, Cdc42 and Rac. With the recent discovery of several novel isoforms, Paks are now categorized into two subgroups based on architectural similarities. The Group I Paks (Pak1, Pak2, Pak3) have been studied in greater detail and shown to be involved in the regulation of cellular processes such as gene transcription, cell morphology, motility, and apoptosis. Here we summarize recent findings that shed light on the newly recognized Group II Paks (Pak4, Pak5, Pak6) and review both similarities and differences between kinases of the two Pak subgroups.

Animals↗

PubMed2002

Detection of peptides, proteins, and drugs that selectively interact with protein targets.

Genome sequencing has been completed for multiple organisms, and pilot proteomic analyses reported for yeast and higher eukaryotes. This work has emphasized the facts that proteins are frequently engaged in multiple interactions, and that governance of protein interaction specificity is a primary means of regulating biological systems. In particular, the ability to deconvolute complex protein interaction networks to identify which interactions govern specific signaling pathways requires the generation of biological tools that allow the distinction of critical from noncritical interactions. We report the application of an enhanced Dual Bait two-hybrid system to allow detection and manipulation of highly specific protein-protein interactions. We summarize the use of this system to detect proteins and peptides that target well-defined specific motifs in larger protein structures, to facilitate rapid identification of specific interactors from a pool of putative interacting proteins obtained in a library screen, and to score specific drug-mediated disruption of protein-protein interaction.

Adaptor Proteins, Signal Transducing↗

PubMed2001

p21-activated kinase links Rac/Cdc42 signaling to merlin.

The neurofibromatosis type 2 tumor suppressor gene, NF2, is mutated in the germ line of NF2 patients and predisposes affected individuals to intracranial and spinal tumors. Moreover, somatic mutations of NF2 can occur in the sporadic counterparts of these neurological tumor types as well as in certain neoplasms of non-neuroectodermal origin, such as malignant mesothelioma and melanoma. NF2 encodes a 595-amino acid protein, merlin, which exhibits significant homology to the ezrin-radixin-moesin family of proteins. However, the mechanism by which merlin exerts its tumor suppressor activity is not well understood. In this investigation, we show that merlin is phosphorylated in response to expression of activated Rac and activated Cdc42 in mammalian cells. Furthermore, we demonstrate that merlin phosphorylation is mediated by p21-activated kinase (Pak), a common downstream target of both Rac and Cdc42. Both in vivo and in vitro kinase assays demonstrated that Pak can directly phosphorylate merlin at serine 518, a site that affects merlin activity and localization. These biochemical investigations provide insights into the regulation of merlin function and establish a framework for elucidating tumorigenic mechanisms involved in neoplasms associated with merlin inactivation.

3T3 Cells↗