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Jeffrey Field

Publications and source records attributed to Jeffrey Field.

12 recordsLinked to original sources
PubMed2006

Opposing roles for Akt1 and Akt2 in Rac/Pak signaling and cell migration.

The Akt/PKB isoforms have different roles in animals, with Akt2 primarily regulating metabolic signaling and Akt1 regulating growth and survival. Here we show distinct roles for Akt1 and Akt2 in mouse embryo fibroblast cell migration and regulation of the cytoskeleton. Akt1-deficient cells responded poorly to platelet-derived growth factor while Akt2-deficient cells had a dramatically enhanced response, resulting in a substantial increase in dorsal ruffling. Swapping domains between Akt1 and Akt2 demonstrated that the N-terminal region containing the pleckstrin homology domain and a linker region distinguishes the two isoforms, while the catalytic domains are interchangeable. Akt2 knock-out cells also migrated faster than wild-type cells, especially through extracellular matrix (ECM), while Akt1 knock-out cells migrated more slowly than wild-type cells. Consistently, Akt2 knock-out cells had elevated Pak1 and Rac activities, suggesting that Akt2 inhibits Rac and Pak1. Both Akt2 and Akt1 associated in complexes with Pak1, but only Akt2 inhibited Pak1 in kinase assays, suggesting an underlying molecular basis for the different cellular phenotypes. Together these data provide evidence for an unexpected functional link between Akt2 and Pak1 that opposes the actions of Akt1 on cell migration.

Animals↗
PubMed2006

Schwann cells: origins and role in axonal maintenance and regeneration.

The Schwann cell plays a vital role in maintaining the peripheral nervous system (PNS). Schwann cells are derived from neural crest cells, and come in two types either myelinating or non-myelinating Schwann cells. Both play a pivotal role in the maintenance and regeneration of axons of the neurons in the PNS. The regulation of Schwann cells is mediated a number of different neurotrophic factors which signal to transcription factors such as Krox-20, Oct-6 and Sox-10. Schwann cells are affected in a number of demyelinating disorders, such as Charcot-Marie-Tooth disease and Guillain-Barré Syndrome, infected by Mycobacterium leprae to cause leprosy and are responsible for the tumors seen in patients with neurofibromatosis type 1 and neurofibromatosis type 2. The Schwann cell is under investigation as a therapeutic agent for demyelinating diseases and spinal cord injuries. Further research on Schwann cells will help understand these diseases and perhaps lead to new treatments.

Axons↗
PubMed2006

cGMP-dependent protein kinase phosphorylates p21-activated kinase (Pak) 1, inhibiting Pak/Nck binding and stimulating Pak/vasodilator-stimulated phosphoprotein association.

Endothelial cells are normally non-motile and quiescent; however, endothelial cells will become permeable and invade and proliferate to form new blood vessels (angiogenesis) in response to wounding, cancer, diabetic retinopathy, age-related macular degeneration, or rheumatoid arthritis. p21-activated kinase (Pak), an effector for the Rho GTPases Rac and Cdc42, is required for angiogenesis and regulates endothelial cell permeability and motility. Although Pak is primarily activated by Rac and Cdc42, there are additional proteins that regulate Pak activity and localization, including three AGC protein kinase family members, Akt-1, PDK-1, and cAMP-dependent protein kinase. We describe phosphorylation and regulation of Pak localization by a fourth AGC kinase family member, cGMP-dependent protein kinase (PKG). Using in vitro mapping, a phosphospecific antibody, co-transfection assays, and untransfected bovine aortic endothelial cells we determined that PKG phosphorylates Pak at serine 21. Phosphorylation was accompanied by changes in proteins associated with Pak. The adaptor protein Nck was released, whereas a novel complex with vasodilator-stimulated phosphoprotein was stimulated. Furthermore Ser-21 phosphorylation of Pak appears to be important for regulation of cell morphology. In both human umbilical vein endothelial cells and HeLa cells, activation of PKG in the presence of Pak stimulated tail retraction and cell polarization. However, in cells expressing S21A mutant Pak1, PKG activation or treatment with a peptide that blocks Nck/Pak binding caused aberrant cell morphology, blocked cell retraction, and mislocalized Pak, producing uropod (tail-like) structures. These data suggest that PKG regulates Pak and that the interaction plays a role in tail retraction.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Comparison of p53 mutations induced by PAH o-quinones with those caused by anti-benzo[a]pyrene diol epoxide in vitro: role of reactive oxygen and biological selection.

Polycyclic aromatic hydrocarbons (PAH) are one of the major carcinogens in tobacco smoke. They are metabolically activated through different routes to form either diol-epoxides, PAH o-quinones, or radical cations, each of which has been proposed to be an ultimate carcinogen. To study how PAH metabolites mutate p53, we used a yeast reporter gene assay based on p53 transcriptional activity. Colonies expressing wt p53 turn white (ADE +) and those expressing mutant p53 turn red (ADE -). We examined the mutagenicity of three o-quinones, benzo[a]pyrene-7,8-dione, benz[a]anthracene-3,4-dione, and dimethylbenz[a]anthracene-3,4-dione, and compared them with (+/-)-anti-benzo[a]pyrene diol epoxide ((+/-)-anti-BPDE) within the same system. The PAH o-quinones tested gave a dose-dependent increase in mutation frequency in the range of 0.160-0.375 microM quinone, provided redox-cycling conditions were used. The dominant mutations were G to T transversions (>42%), and the incidence of hotspot mutations in the DNA-binding domain was more than twice than that expected by a random distribution. The dependence of G to T transversions on redox cycling implicates 8-oxo-dGuo as the lesion responsible, which is produced under identical conditions (Chem. Res. Toxicol. (2005) 18, 1027). A dose-dependent mutation frequency was also observed with (+/-)-anti-BPDE but at micromolar concentrations (0-20 microM). The mutation pattern observed was G to C (63%) > G to A (18%) > G to T (15%) in umethylated p53 and was G to A (39%) > G to C (34%) > G to T (16%) in methylated p53. The preponderance of G mutations is consistent with the formation of anti-BPDE-N2-dGuo as the major adduct. The frequency of hotspots mutated by (+/-)-anti-BPDE was essentially random in umethylated and methylated p53, suggesting that 5'-CpG-3' islands did not direct mutations in the assay. These data suggest that smoking may cause mutations in p53 by formation of PAH o-quinones, which produce reactive oxygen species. The resultant 8-oxo-dGuo yields a pattern of mutations but not a spectrum consistent with that seen in lung cancer; we suggest that the emergence of the spectrum requires biological selection.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2005

p21-activated Kinase 1 (Pak1)-dependent phosphorylation of Raf-1 regulates its mitochondrial localization, phosphorylation of BAD, and Bcl-2 association.

Raf-1 protects cells from apoptosis, independently of its signals to MEK and ERK, by translocating to the mitochondria where it binds Bcl-2 and displaces BAD. However, the answer to the question of how Raf-1 is normally lured to the mitochondria and becomes activated remains elusive. p21-activated protein kinases (Paks) are serine/threonine protein kinases that phosphorylate Raf-1 at Ser-338 and Ser-339. Here we elucidate the molecular mechanism through which Pak1 signals to BAD through a Raf-1-activated pathway. Upon phosphorylation by Pak1, Raf-1 translocates to mitochondria and phosphorylates BAD at Ser-112. Moreover, the mitochondrial translocation of Raf-1 and the interaction between Raf-1 and Bcl-2 are regulated by Raf-1 phosphorylation at Ser-338/Ser-339. Notably, we show that formation of a Raf-1-Bcl-2 complex coincides with loss of an interaction between Bcl-2 and BAD. These signals are specific for Pak1, because Src-activated Raf-1 only stimulates the MAP kinase cascade. Thus, our data identify the molecular connections of a Pak1-Raf-1-BAD pathway that is involved in cell survival signaling.

Animals↗
PubMed2005

Rho, Rac, Pak and angiogenesis: old roles and newly identified responsibilities in endothelial cells.

Angiogenesis-the develoment of microvasculature-requires, in part, directed endothelial cell motility and responsiveness to external signals. Several of the proteins, which modulate and/or direct endothelial cell motility and morphology in angiogenesis are the Rho GTPases (Rho, Rac, and Cdc42) and Pak (a downstream effector of Rac and Cdc42). Previously, overexpression and activation of Rho GTPases and Pak had been implicated in the development of cancer, through their roles in cancer cell transformation, stimulation of proliferation, inhibition of apoptosis, and migration. Yet regardless of the transformed status of cells within a tumor, without a blood supply most tumors cannot grow larger than 1-2 mm. The blood supply in tumors is provided by capillaries formed of endothelial cells in a process called angiogenesis. Consequently, there is enormous interest in the role of the wild type endothelial cells-and the signaling mechanisms required to support angiogenesis and subsequent growth of metastatic and aggressive cancers. Recent work has begun to uncover the roles of the Rho GTPases and Pak in the regulation of normal endothelial cell function. This review will discuss the current literature regarding the roles of Rho and Rac, and the Rac effector-Pak, in endothelial cells, and we will propose new avenues of research for interaction of the AGC kinase-PKG, with the Rho GTPases and Pak in the cell motility and cell morphology of endothelial cells.

Cell Movement↗
PubMed2004

c-Abl phosphorylates Dok1 to promote filopodia during cell spreading.

Filopodia are dynamic F-actin structures that cells use to explore their environment. c-Abl tyrosine kinase promotes filopodia during cell spreading through an unknown mechanism that does not require Cdc42 activity. Using an unbiased approach, we identified Dok1 as a specific c-Abl substrate in spreading fibroblasts. When activated by cell adhesion, c-Abl phosphorylates Y361 of Dok1, promoting its association with the Src homology 2 domain (SH2)/SH3 adaptor protein Nck. Each signaling component was critical for filopodia formation during cell spreading, as evidenced by the finding that mouse fibroblasts lacking c-Abl, Dok1, or Nck had fewer filopodia than cells reexpressing the product of the disrupted gene. Dok1 and c-Abl stimulated filopodia in a mutually interdependent manner, indicating that they function in the same signaling pathway. Dok1 and c-Abl were both detected in filopodia of spreading cells, and therefore may act locally to modulate actin. Our data suggest a novel pathway by which c-Abl transduces signals to the actin cytoskeleton through phosphorylating Dok1 Y361 and recruiting Nck.

Actins↗
PubMed2003

Activation of p21-activated kinase 1-nuclear factor kappaB signaling by Kaposi's sarcoma-associated herpes virus G protein-coupled receptor during cellular transformation.

Kaposi's sarcoma-associated herpes virus (KSHV) contributes to the pathogenesis of Kaposi's sarcoma and primary effusion lymphomas. KSHV encodes a G protein-coupled receptor (KSHV-GPCR) that signals constitutively and transforms NIH3T3 cells. Here, we show that KSHV-GPCR transformation requires activation of the small G protein Rac1 and its effector, the p21-activated kinase 1 (Pak1). Either transient or sustained expression of KSHV-GPCR activated both Rac1 and Pak1. Furthermore, expression of dominant-negative mutants of Rac (RacN17) or Pak1 (PakR299, Pak-PID) inhibited KSHV-GPCR-induced focus formation and growth in soft agar. We also demonstrate that signaling from Pak1 to nuclear factor-kappaB (NFkappaB) is required for cell transformation induced by KSHV-GPCR. KSHV-GPCR induced transcriptional activation by NFkappaB. This process is inhibited by the PAK-PID, whereas reciprocally, expression of constitutively active Pak1 (PakL107F) activated NFkappaB comparably to KSHV-GPCR. The Pak-PID and RacN17 inhibited the KSHV-GPCR-induced phosphorylation of inhibitor of kappaB kinase-beta and inhibitor of kappaB-alpha, implying that it is Pak1-dependent phosphorylation and subsequent destruction of the inhibitor of kappaB proteins that allows NFkappaB activation. Finally, experiments with the KSHV-GPCR inverse agonist interferon-gamma-inducible protein-10, the Galpha(i) inhibitor pertussis toxin, and an inhibitor of phosphatidylinositol 3'-kinase, wortmannin, indicate that signaling through the Galpha(i) pathway and phosphatidylinositol 3'-kinase contributes to the cell transformation and NFkappaB activation induced by the KSHV-GPCR.

Animals↗
PubMed2003

Lysophosphatidic acid promotes survival and differentiation of rat Schwann cells.

Lysophosphatidic acid (LPA; 1-acyl-sn-glycerol-3-phosphate), an abundant constituent of serum, mediates multiple biological responses via G protein-coupled serpentine receptors. Schwann cells express the LPA receptors (Edg receptors), which, once activated, have the potential to signal through G(alphai) to activate p21(ras) and phosphatidylinositol 3-kinase, through G(alphaq) to activate phospholipase C, or through G(q12/13) to activate the Rho pathway. We found that the addition of serum or LPA to serum-starved Schwann cells rapidly (10 min) induced the appearance of actin stress fibers via a Rho-mediated pathway. Furthermore, LPA was able to rescue Schwann cells from apoptosis in a G(alphai)/phosphatidylinositol 3-kinase/MEK/MAPK-dependent manner. In addition, LPA increased the expression of myelin protein P(0) in Schwann cells in a Galpha(i)-independent manner but dependent on protein kinase C. By means of pharmacological and overexpression approaches, we found that the novel isozyme protein kinase Cdelta was required for myelin P(0) expression. Thus, the multiple effects of LPA in Schwann cells (actin reorganization, survival, and myelin gene expression) appear to be mediated through the different G protein-dependent pathways activated by the LPA receptor.

Adenoviridae↗
PubMed2003

Akt phosphorylation of serine 21 on Pak1 modulates Nck binding and cell migration.

The p21-activated protein kinases (Paks) regulate cellular proliferation, differentiation, transformation, and survival through multiple downstream signals. Paks are activated directly by the small GTPases Rac and Cdc42 and several protein kinases including Akt and PDK-1. We found that Akt phosphorylated and modestly activated Pak1 in vitro. The major site phosphorylated by Akt on Pak1 mapped to serine 21, a site originally shown to be weakly autophosphorylated on Pak1 when Cdc42 or Rac activates it. A peptide derived from the region surrounding serine 21 was a substrate for Akt but not Pak1 in vitro, and Akt stimulated serine 21 phosphorylation on the full-length Pak1 much better than Rac did. The adaptor protein Nck binds Pak near serine 21, and its association is regulated by phosphorylation of this site. We found that either treatment of Pak1 in vitro with Akt or coexpression of constitutively active Akt with Pak1 reduced Nck binding to Pak1. In HeLa cells, green fluorescent protein-tagged Pak1 was concentrated at focal adhesions and was released when Akt was cotransfected. A peptide containing the Nck binding site of Pak1 fused to a portion of human immunodeficiency virus Tat to allow it to enter cells was used to test the functional importance of Nck/Pak binding in Akt-stimulated cell migration. This Tat-Nck peptide reduced Akt-stimulated cell migration. Together, these data suggest that Akt modulates the association of Pak with Nck to regulate cell migration.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Reactive oxygen species generated by PAH o-quinones cause change-in-function mutations in p53.

Polycyclic aromatic hydrocarbons (PAHs) in tobacco smoke may cause human lung cancer via metabolic activation to ultimate carcinogens. p53 is one of the most commonly mutated tumor suppressor genes in this disease. An analysis of the p53 mutational database shows that G to T transversions are a signature mutation of lung cancer. Aldo-keto reductases (AKRs) activate PAH trans-dihydrodiol proximate carcinogens to yield their corresponding reactive and redox-active o-quinones, e.g., benzo[a]pyrene-7,8-dione (BP-7,8-dione). We employed a yeast reporter system to determine whether PAH o-quinones or the ROS they generate cause change-in-function mutations in p53. N-Methyl-N-nitroso-N'-nitro-guanidine, a standard alkylating mutagen was used as a positive control. MNNG caused a dose-dependent increase in mutant yeast colonies and at the highest concentrations 8-14% of the yeast colonies were mutated and were characterized by G:C to A:T transitions in the p53 DNA binding domain. Treatment of p53 cDNA with micromolar concentrations of (+/-)-anti-7,8-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene, (anti-BPDE, an ultimate carcinogen) or sub-micromolar concentrations of BP-7,8-dione in the presence of redox-cycling conditions (NADPH and CuCl(2)) also caused p53 mutations in a dose-dependent manner. We found that no mutants were observed with PAH o-quinones or NADPH alone. p53 mutagenesis by BP-7,8-dione was attenuated by ROS scavengers and completely abrogated by a combination of superoxide dismutase and catalase, indicating that both superoxide anion and hydroxyl radicals were the responsible mutagens. The bulk of the mutations detected were single-point mutations and were not random in occurrence. Over 46% of BP-7,8-dione-induced mutations were G:C to T:A transversions, consistent with the formation of 8-oxo-dGuo or its secondary oxidation products. In addition, 25% of these mutations were at hotspots in p53 which are known to be mutated in lung cancer. Together these data suggest that PAH o-quinones generate an endogenous mutagen (ROS) which leads to p53 inactivation. These observations provide an alternative route to G to T transversions that dominate in p53 in lung cancer.

Alcohol Oxidoreductases↗