All nucleoside reverse transcriptase inhibitors (NRTI) must first be metabolized to their triphosphate forms in order to be active against HIV. Zidovudine (ZDV), abacavir (ABC) and lamivudine (3TC) ...have proven to be an efficacious combination. In order simultaneously to measure intracellular levels of the triphosphates (-TP) of ZDV, ABC (carbovir, CBV) and 3TC, either together or individually, we have developed a cartridge-LC–MS/MS method. The quantitation range was 2.5–250
pg/μl for 3TC-TP, 0.1–10.0
pg/μl for ZDV-TP and 0.05–5.00
pg/μl for CBV-TP. This corresponds to 0.1–11.0
pmol 3TC-TP per million cells, 4–375
fmol ZDV-TP per million cells and 2–200
fmol CBV-TP per million cells, extracted from 10 million cells. Patient samples demonstrated measured levels in the middle regions of our standard curves both at pre-dose and 4
h post-dose times.
We sought to characterize the role of upstream kinases in the regulation of the MAP3 kinase MEKK1 and the potential impact
on signaling to MAP kinase cascades. We find that the MAP4 kinase PAK1 ...phosphorylates the amino terminus of MEKK1 on serine
67. We show that serine 67 lies in a D domain, which binds to the c-Jun-NH 2 -terminal kinase/stress-activated protein kinases (JNK/SAPK). Serine 67 is constitutively phosphorylated in resting 293 cells,
but is dephosphorylated following exposure to stress stimuli such as anisomycin and UV irradiation. Phosphorylation of this
site inhibits binding of JNK/SAPK to MEKK1. Thus, we propose a mechanism by which the MEKK1-dependent JNK/SAPK pathway is
negatively regulated by PAK through phosphorylation of serine 67.
RGS4 and RGS10 expressed in Sf9 cells are palmitoylated at a conserved Cys residue (Cys 95 in RGS4, Cys 66 in RGS10) in the regulator of G protein signaling (RGS) domain that is also ...autopalmitoylated when the purified proteins
are incubated with palmitoyl-CoA. RGS4 also autopalmitoylates at a previously identified cellular palmitoylation site, either
Cys 2 or Cys 12 . The C2A/C12A mutation essentially eliminates both autopalmitoylation and cellular 3 Hpalmitate labeling of Cys 95 . Membrane-bound RGS4 is palmitoylated both at Cys 95 and Cys 2/12 , but cytosolic RGS4 is not palmitoylated. RGS4 and RGS10 are GTPase-activating proteins (GAPs) for the G i and G q families of G proteins. Palmitoylation of Cys 95 on RGS4 or Cys 66 on RGS10 inhibits GAP activity 80â100% toward either Gα i or Gα z in a single-turnover, solution-based assay. In contrast, when GAP activity was assayed as acceleration of steady-state GTPase
in receptor-G protein proteoliposomes, palmitoylation of RGS10 potentiated GAP activity â¥20-fold. Palmitoylation near the
N terminus of C95V RGS4 did not alter GAP activity toward soluble Gα z and increased G z GAP activity about 2-fold in the vesicle-based assay. Dual palmitoylation of wild-type RGS4 remained inhibitory. RGS protein
palmitoylation is thus multi-site, complex in its control, and either inhibitory or stimulatory depending on the RGS protein
and its sites of palmitoylation.
The T cell receptor (TCR) zeta subunit contains three immunoreceptor tyrosine-based activation motifs (ITAMs) that translate effective extracellular ligand binding into intracellular signals by ...becoming phosphorylated into 21- and 23-kD forms. We report here that the 21-kD form of TCR zeta is generated by phosphorylation of the tyrosines in the second and third ITAMs, whereas the 23-kD form is formed by the additional phosphorylation of the membrane-proximal ITAM tyrosines. The stable formation of the 21- and 23-kD species requires the binding of the tandem SH2 domains of ZAP-70. We also report that TCR-mediated signaling processes can proceed independently of either the 21- or 23-kD species of TCR zeta.
Article Watch: April 2016 Slaughter, Clive A.
Journal of biomolecular techniques,
04/2016, Volume:
27, Issue:
1
Journal Article
Peer reviewed
Open access
This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and ...useful to Clive Slaughter, MCG-UGA Medical Partnership, 1425 Prince Ave., Athens, GA 30606, USA; Tel: (706) 713-2216; Fax: (706) 713-2221; E-mail: cslaught@uga.edu, or to any member of the editorial board. Article summaries reflect the reviewer’s opinions and not necessarily those of the association.
Article Watch: December 2015 Slaughter, Clive A.
Journal of biomolecular techniques,
12/2015, Volume:
26, Issue:
4
Journal Article
Peer reviewed
Open access
This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and ...useful to: Clive Slaughter, GRU-UGA Medical Partnership, 1425 Prince Ave., Athens, GA 30606, USA. Tel: (706) 713-2216; Fax: (706) 713-2221; E-mail: cslaught@uga.edu; or to any member of the editorial board. Article summaries reflect the reviewer’s opinions and not necessarily those of the association.
PDF
