Replication of HIV-1 Nef chimeras in U87MG-CD4⁺/CXCR4⁺ cells.

<p>A) U87MG- CD4⁺/CXCR4⁺ cells (2×10⁴ per well of a 96-well plate) were infected with 200 pg p24 equivalents/ml of wild-type HIV-1 NL4-3, a Nef-defective mutant (ΔNef), or the indicated Nef chimeras in a final culture volume of 200 µl. HIV p24 levels were determined by ELISA 5 days later. Data are presented as percent of p24 release observed relative to the HIV-1 NL4-3 control ± S.D. B) U87MG-CD4⁺CXCR4⁺ cells (1×10⁵ per well of a 6-well plate) were infected with 1 ng p24 equivalents/ml of the same panel of viruses as in part A. Viral Nef and gag protein expression was verified by immunoblotting lysates of infected cells 4 days later. Lysates from uninfected cells are included as a negative control (U87MG; far left lane).</p

Sequence alignment of primary and laboratory Nef alleles used in this study.

<p>Nef cDNA clones representative of all major non-recombinant HIV-1 clades were selected from the NIH HIV-1 sequence database as described in the text. The first letter in each clone ID (bold) indicates the subtype assignment. This Clustal W alignment also includes Nef-SF2 (top), which has been studied extensively in our laboratory in terms of Hck activation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032561#pone.0032561-Briggs1" target="_blank">[41]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032561#pone.0032561-Lerner1" target="_blank">[43]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032561#pone.0032561-Trible1" target="_blank">[44]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032561#pone.0032561-Choi1" target="_blank">[50]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032561#pone.0032561-EmertSedlak1" target="_blank">[53]</a>, as well as Nef-ELI, which fails to activate Hck or other SFKs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032561#pone.0032561-Trible1" target="_blank">[44]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032561#pone.0032561-Choi1" target="_blank">[50]</a>. Key residues in the SF2 sequence that are essential for SH3 binding are shown in red, and include the PxxPxR motif and hydrophobic pocket residues F90, W113, and Y/F120. In Nef-ELI, Y120 is replaced with isoleucine (highlighted in yellow); this single substitution accounts for loss of SH3 engagement and Hck activation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032561#pone.0032561-Choi1" target="_blank">[50]</a>. Residues involved in Nef dimerization include K/R105, L112, Y115, F121, and D123 (blue). Flanking N- and C-terminal Nef sequences are more variable and have been omitted for clarity.</p

Diphenylfuropyrimidine (DFP) analogs are broadly active against primary Nef-mediated Hck activation.

<p>A) Structures of 4-amino DFP (DFP-4A) and the corresponding 4-aminopropanol (DFP-4AP) and 4-aminobutanol (DFP-4AB) analogs. B) The activity of recombinant downregulated Hck (Hck-YEEI) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032561#pone.0032561-Trible1" target="_blank">[44]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032561#pone.0032561-Trible2" target="_blank">[52]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032561#pone.0032561-EmertSedlak1" target="_blank">[53]</a> was determined in the absence or presence of the indicated Nef proteins as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032561#s3" target="_blank">Materials and Methods</a>. Reactions were run in the presence of 10 µM concentrations of DFP-4A, DFP-4AP, DFP-4AB, or the DMSO carrier solvent as negative control. Results are expressed as the mean percent of maximum substrate phosphorylation ± S.D.; this experiment was repeated twice with comparable results.</p

Primary M-group HIV-1 Nef proteins strongly activate Lyn in cells.

<p>Yeast cultures were transformed with a galactose-inducible expression plasmid for Lyn-YEEI, which carries a modified C-terminal tail that enables downregulation in the absence of Csk <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032561#pone.0032561-Trible2" target="_blank">[52]</a>, or the empty expression plasmid as a negative control (Con). Where indicated, Lyn-YEEI cells were co-transformed with galactose-inducible vectors for Nef-ELI, Nef-SF2, and ten primary Nef alleles (A1, A2, B, C, F1, F2, G, H, J, and K). Transformed cells were grown in liquid culture in the presence of galactose at 30°C for 18 h. Protein extracts were separated via SDS-PAGE and immunoblotted for tyrosine-phosphorylated proteins (<i>pTyr</i>) as well as for Nef, Lyn and actin as a loading control. Nef-ELI is unable to interact with Lyn and serves as an additional negative control <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032561#pone.0032561-Trible2" target="_blank">[52]</a>. This experiment was repeated three times with comparable results.</p

Structural features of the Nef:SH3 and Nef dimerization interfaces.

<p>A) Overview of the Nef:SH3 dimer X-ray crystal structure, based on the crystal coordinates of Lee, et al. (PDB: 1EFN) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032561#pone.0032561-Lee1" target="_blank">[48]</a>. The monomeric Nef core subunits are modeled in blue and green respectively; the SH3 domains are shown in red. B) Close-up view of the Nef:SH3 interface. Nef residues P72 and P75 define a polyproline type II helix that meshes with the hydrophobic grooves of the SH3 surface, and is oriented and stabilized by an ionic interaction between Nef R77 and SH3 D100. High affinity interaction also requires a hydrophobic pocket formed in part by Nef residues F90, W113, and Y120. This pocket engages SH3 domain RT loop I96 (Van der Waals surface shown as dots). Mutagenesis of either Nef Y120 or SH3 I96 is sufficient to disrupt Nef:SH3 interaction <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032561#pone.0032561-Choi1" target="_blank">[50]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032561#pone.0032561-Lee2" target="_blank">[66]</a>. C) Close-up view of the Nef dimerization interface. Dimer packing involves hydrophobic interactions of side chains of the αB helices (L112, Y115, F121) which are orthogonally opposed. This hydrophobic core is capped on both ends by ionic interactions involving D123 and R105.</p

Inhibition of HIV-1 Nef chimera-mediated activation of SFKs by 4-amino DFP analogs.

<p>CEM-T4 cells were infected with wild-type HIV-1 NL4-3 (WT), a Nef-defective mutant (ΔNef), or the indicated Nef chimeras in the absence or presence of 3 µM DFP-4A, DFP-4AP, DFP-4AB, or the carrier solvent (DMSO) alone as a control (Con). SFK proteins were immunoprecipitated from infected cell lysates followed by immunoblotting with a phosphospecific antibody against the activation loop phosphotyrosine residue common to all SFKs (pY418). Control blots were performed on cell lysates for HIV-1 Gag proteins (p55, p40, and p24), Nef, as well as actin as a loading control. Results from uninfected cells are shown in the far right lane (No virus). This experiment was repeated twice with comparable results.</p

Purified primary Nef proteins bind to the Hck SH3 domain and activate downregulated Hck in vitro.

<p>A) SDS-PAGE of recombinant purified proteins. Each of the indicated Nef subtypes were expressed in bacteria and purified with N-terminal His-tags (<i>left</i>). GST and GST fusion proteins with the Hck SH3 domain (wild-type and W93A mutant) were also expressed in bacteria and immobilized on glutathione-agarose beads (<i>right</i>). Protein aliquots were resolved by SDS-PAGE and stained with Coomassie blue. B) Nef-SH3 binding assay. Recombinant His-tagged Nef proteins (1 µg) were incubated with equimolar amounts of immobilized GST and GST-SH3 fusion proteins. The agarose beads were then washed, and associated Nef proteins were detected by immunoblotting using antibodies to the His-tags. C) Nef-induced Hck activation. Kinase activity of recombinant downregulated Hck (Hck-YEEI; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032561#pone.0032561-Trible1" target="_blank">[44]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032561#pone.0032561-Trible2" target="_blank">[52]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032561#pone.0032561-EmertSedlak1" target="_blank">[53]</a> was determined in the absence or presence of the indicated Nef proteins using the Z-Lyte assay as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032561#s3" target="_blank">Materials and Methods</a>. Results are expressed as the mean percent of maximum substrate phosphorylation ± S.D.; this experiment was repeated twice with comparable results.</p

Primary M-group HIV-1 Nef proteins strongly activate Hck in cells.

<p>Yeast cultures were transformed with a galactose-inducible expression plasmid for Hck-YEEI, which carries a modified C-terminal tail that enables downregulation in the absence of Csk <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032561#pone.0032561-Trible2" target="_blank">[52]</a>, or the empty expression plasmid as a negative control (Con). Where indicated, Hck-YEEI cells were co-transformed with galactose-inducible vectors for Nef-ELI, Nef-SF2, and ten primary Nef alleles (A1, A2, B, C, F1, F2, G, H, J, and K). Transformed cells were grown in liquid culture in the presence of galactose at 30°C for 18 h. Protein extracts were separated via SDS-PAGE and immunoblotted for tyrosine-phosphorylated proteins (<i>pTyr</i>) as well as for Nef, Hck and actin as a loading control. Nef-ELI is unable to interact with Hck and serves as an additional negative control <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032561#pone.0032561-Trible2" target="_blank">[52]</a>. This experiment was repeated three times with comparable results.</p

c‑Yes Tyrosine Kinase Is a Potent Suppressor of ES Cell Differentiation and Antagonizes the Actions of Its Closest Phylogenetic Relative, c‑Src

Embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst stage embryo and are characterized by self-renewal and pluripotency. Previous work has shown that Src-family tyrosine kinases display dynamic expression and activity changes during ES cell differentiation, suggesting distinct functions in the control of developmental fate. Here we used ES cells to test the hypothesis that c-Src and its closest phylogenetic relative, c-Yes, act in biological opposition despite their strong homology. Unlike c-Src, enforced expression of active c-Yes blocked ES cell differentiation to embryoid bodies by maintaining pluripotency gene expression. To explore the interplay of c-Src and c-Yes in ES cell differentiation, we engineered c-Src and c-Yes mutants that are resistant to A-419259, a potent pyrrolopyrimidine inhibitor of the Src kinase family. Previous studies have shown that A-419259 treatment blocks all Src-family kinase activity in ES cells, preventing differentiation while maintaining pluripotency. Expression of inhibitor-resistant c-Src but not c-Yes rescued the A-419259 differentiation block, resulting in a cell population with properties of both primitive ectoderm and endoderm. Remarkably, when inhibitor-resistant c-Src and c-Yes were expressed together in ES cells, c-Yes activity suppressed c-Src-mediated differentiation. These studies show that even closely related kinases such as c-Src and c-Yes have unique and opposing functions in the same cell type. Selective agonists or inhibitors of c-Src versus c-Yes activity may allow more precise pharmacological manipulation of ES cell fate and have broader applications in other biological systems that express multiple Src family members such as tumor cells

Tim is a c-Src SH3 domain binding protein and substrate.

<p><b>A</b>, Lysates were prepared from ES cells and EBs and incubated with immobilized GST, the Src GST-SH3 fusion protein, or the corresponding inactive GST-SH3 mutant. Following washing, bound proteins were separated by SDS-PAGE, transferred to PVDF membranes and probed with an anti-peptide antibody to Tim. Full-length Tim and a discrete cleavage product (CP) were found to associate with the GST-SH3 fusion protein, but not with GST alone or with the mutant GST-SH3 domain. <b>B</b>, Tim is a substrate for c-Src. Human 293T cells were transfected wild-type c-Src (Src-WT), a kinase-defective mutant (Src-KD), or with the empty expression plasmid (Con) together with V5 epitope-tagged mouse Tim as indicated. Tim was immunoprecipitated from the transfected cell lysates with a V5 antibody and immunoblotted for Tim protein recovery (Tim), tyrosine phosphorylation (pTyr), and ubiquitin (Ub). Tranfected Src protein expression was confirmed in the cell lysates, with actin as a loading control.</p