Chemical library screens targeting an HIV-1 accessory factor/host cell kinase complex identify novel antiretroviral compounds.

Nef is an HIV-1 accessory protein essential for AIDS progression and an attractive target for drug discovery. Lack of a catalytic function makes Nef difficult to assay in chemical library screens. We developed a high-throughput screening assay for inhibitors of Nef function by coupling it to one of its host cell binding partners, the Src-family kinase Hck. Hck activation is dependent upon Nef in this assay, providing a direct readout of Nef activity in vitro. Using this screen, a unique diphenylfuropyrimidine was identified as a strong inhibitor of Nef-dependent Hck activation. This compound also exhibited remarkable antiretroviral effects, blocking Nef-dependent HIV replication in cell culture. Structurally related analogs were synthesized and shown to exhibit similar Nef-dependent antiviral activity, identifying the diphenylfuropyrimidine substructure as a new lead for antiretroviral drug development. This study demonstrates that coupling noncatalytic HIV accessory factors with host cell target proteins addressable by high-throughput assays may afford new avenues for the discovery of anti-HIV agents.

HGF and c-Met participate in paracrine tumorigenic pathways in head and neck squamous cell cancer.

PURPOSE: We determined hepatocyte growth factor (HGF) and c-Met expression and signaling in human head and neck squamous cell carcinoma (HNSCC) cells and primary tissues and tested the ability of c-Met tyrosine kinase inhibitors (TKI) to block HGF-induced biological signaling. EXPERIMENTAL DESIGN: Expression and signaling were determined using immunoblotting, ELISA, and immunohistochemistry. Biological end points included wound healing, cell proliferation, and invasion. c-Met TKIs were tested for their ability to block HGF-induced signaling and biological effects in vitro and in xenografts established in nude mice. RESULTS: c-Met was expressed and functional in HNSCC cells. HGF was secreted by HNSCC tumor-derived fibroblasts, but not by HNSCC cells. Activation of c-Met promoted phosphorylation of AKT and mitogen-activated protein kinase as well as release of the inflammatory cytokine interleukin-8. Cell growth and wound healing were also stimulated by HGF. c-Met TKIs blocked HGF-induced signaling, interleukin-8 release, and wound healing. Enhanced invasion of HNSCC cells induced by the presence of tumor-derived fibroblasts was completely blocked with a HGF-neutralizing antibody. PF-2341066, a c-Met TKI, caused a 50% inhibition of HNSCC tumor growth in vivo with decreased proliferation and increased apoptosis within the tumors. In HNSCC tumor tissues, both HGF and c-Met protein were increased compared with expression in normal mucosa. CONCLUSIONS: These results show that HGF acts mainly as a paracrine factor in HNSCC cells, the HGF/c-Met pathway is frequently up-regulated and functional in HNSCC, and a clinically relevant c-Met TKI shows antitumor activity in vivo. Blocking the HGF/c-Met pathway may be clinically useful for the treatment of HNSCC.

Functional characterization and conformational analysis of the Herpesvirus saimiri Tip-C484 protein.

Tyrosine kinase interacting protein (Tip) of Herpesvirus saimiri (HVS) activates the lymphoid-specific member of the Src family kinase Lck. The Tip:Lck interaction is essential for transformation and oncogenesis in HVS-infected cells. As there are no structural data for Tip, hydrogen-exchange mass spectrometry was used to investigate the conformation of a nearly full-length form (residues 1-187) of Tip from HVS strain C484. Disorder predictions suggested that Tip would be mostly unstructured, so great care was taken to ascertain whether recombinant Tip was functional. Circular dichroism and gel-filtration analysis indicated an extended, unstructured protein. In vitro and in vivo binding and kinase assays confirmed that purified, recombinant Tip interacted with Lck, was capable of activating Lck kinase activity strongly and was multiply phosphorylated by Lck. Hydrogen-exchange mass spectrometry of Tip then showed that the majority of backbone amide hydrogen atoms became deuterated after only 10 s of labeling. Such a result suggested that Tip was almost totally unstructured in solution. Digestion of deuterium-labeled Tip revealed some regions with minor protection from exchange. Overall, it was found that, although recombinant Tip is still functional and capable of binding and activating its target Lck, it is largely unstructured.

C-terminal Src kinase-homologous kinase (CHK), a unique inhibitor inactivating multiple active conformations of Src family tyrosine kinases.

The Src family of protein kinases (SFKs) mediates mitogenic signal transduction, and constitutive SFK activation is associated with tumorigenesis. To prevent constitutive SFK activation, the catalytic activity of SFKs in normal mammalian cells is suppressed mainly by two inhibitors called C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK), which inactivate SFKs by phosphorylating a consensus tyrosine near the C terminus of SFKs (Y(T)). The phosphorylated Y(T) intramolecularly binds to the SH2 domain of SFKs. This interaction, known as pY(T)/SH2 interaction, together with binding between the SH2 kinase linker and the SH3 domain of SFKs (linker/SH3 interaction) stabilizes SFKs in a "closed" inactive conformation. We previously discovered an alternative mechanism CHK employs to inhibit SFKs. This mechanism, referred to as the non-catalytic inhibitory mechanism, involves tight binding of CHK to SFKs; the binding alone is sufficient to inhibit SFKs. Herein, we constructed multiple active conformations of an SFK member, Hck, by systematically disrupting the two inhibitory interactions. We found that CHK employs the non-catalytic mechanism to inactivate these active conformations of Hck. However, CHK does not bind Hck when it adopts the inactive conformation in which both inhibitory interactions are intact. These data indicate that binding of CHK to SFKs via the non-catalytic mechanism is governed by the conformations of SFKs. Although CSK is also an inhibitor of SFKs, it does not inhibit SFKs by a similar non-catalytic mechanism. Thus, the non-catalytic inhibitory mechanism is a unique property of CHK that allows it to down-regulate multiple active conformations of SFKs.

Conformational features of the full-length HIV and SIV Nef proteins determined by mass spectrometry.

The Nef protein from human or simian immunodeficiency virus enhances viral replication, downregulates immune cell receptors, and activates multiple host cell signaling pathways. Conformational information about full-length Nef has been difficult to obtain as the full-length protein is not readily amenable to NMR or X-ray crystallography due to aggregation at high concentrations. As an alternative, full-length HIV and SIV Nef were probed with hydrogen exchange mass spectrometry, a method compatible with the low concentration requirements of Nef. The results showed that HIV Nef contains a solvent-protected core, as previously demonstrated with both NMR and X-ray crystallography. SIV Nef, for which there is no structural information, had a similar protected core, although it was more flexible and dynamic than its HIV counterpart. Many of the regions outside the core in both SIV and HIV Nef were highly solvent exposed. However, limited protection from exchange was observed in both N- and C-terminal regions, suggesting the presence of structured elements. Protection from exchange was also observed in a large loop emanating from the core that was deleted for NMR and X-ray analysis. These data show that while the majority of Nef was highly solvent exposed, regions outside the core may have structural attributes which may contribute to Nef functions known to map to these regions.

Phosphorylation of the varicella-zoster virus (VZV) major transcriptional regulatory protein IE62 by the VZV open reading frame 66 protein kinase.

IE62, the major transcriptional regulatory protein encoded by varicella-zoster virus (VZV), is nuclear at early times of VZV infection but then becomes predominantly cytoplasmic as a result of expression of the protein kinase encoded by open reading frame 66 (ORF66). Cytoplasmic forms of IE62 are required for its inclusion as an abundant VZV virion tegument protein. Here we show that ORF66 directly phosphorylates IE62 at two residues, with phosphorylation at S686 being sufficient to regulate IE62 nuclear import. Phosphotryptic peptide analyses established an ORF66 kinase-mediated phosphorylation of the complete IE62 protein in transfected and VZV-infected cells. Using truncated and point-mutated IE62 peptides, ORF66-directed phosphorylation was mapped to residues S686 and S722, immediately downstream of the IE62 nuclear localization signal. An IE62 protein with an S686A mutation retained efficient nuclear import activity, even in the presence of functional ORF66 protein kinase, but an IE62 protein containing an S686D alteration was imported into the nucleus inefficiently. In contrast, the nuclear import of IE62 carrying an S722A mutation was still modulated by ORF66 expression, and IE62 with an S722D mutation was imported efficiently into the nucleus. An in vitro phosphorylation assay was developed using bacterially expressed IE62-maltose binding protein fusions as substrates for immunopurified ORF66 protein kinase from recombinant baculovirus-infected insect cells. ORF66 kinase phosphorylated the IE62 peptides, with similar specificities for residues S686 and S722. These results indicate that IE62 nuclear import is modulated as a result of direct phosphorylation of IE62 by ORF66 kinase. This represents an interaction that is, so far, unique among the alphaherpesviruses.

An examination of dynamics crosstalk between SH2 and SH3 domains by hydrogen/deuterium exchange and mass spectrometry.

The ability of proteins to regulate their own enzymatic activity can be facilitated by changes in structure or protein dynamics in response to external regulators. Because many proteins contain SH2 and SH3 domains, transmission of information between the domains is a potential method of allosteric regulation. To determine if ligand binding to one modular domain may alter structural dynamics in an adjacent domain, allowing potential transmission of information through the protein, we used hydrogen exchange and mass spectrometry to measure changes in protein dynamics in the SH3 and SH2 domains of hematopoietic cell kinase (Hck). Ligand binding to either domain had little or no effect on hydrogen exchange in the adjacent domain, suggesting that changes in protein structure or dynamics are not a means of SH2/SH3 crosstalk. Furthermore, ligands of varying affinity covalently attached to SH3/SH2 altered dynamics only in the domain to which they bind. Such results demonstrate that ligand binding may not structurally alter adjacent SH3/SH2 domains and implies that other aspects of protein architecture contribute to the multiple levels of regulation in proteins containing SH3 and SH2 domains.

Activation of the Src family kinase Hck without SH3-linker release.

Src family protein-tyrosine kinases are regulated by intramolecular binding of the SH2 domain to the C-terminal tail and association of the SH3 domain with the SH2 kinase-linker. The presence of two regulatory interactions raises the question of whether disruption of both is required for kinase activation. To address this question, we engineered a high affinity linker (HAL) mutant of the Src family member Hck in which an optimal SH3 ligand was substituted for the natural linker. Surface plasmon resonance analysis demonstrated tight intramolecular binding of the modified HAL sequence to SH3. Hck-HAL was then combined with a tail tyrosine mutation (Y501F) and expressed in Rat-2 fibroblasts. Surprisingly, Hck-HAL-Y501F showed strong transforming and kinase activities, demonstrating that intramolecular SH3-linker release is not required for SH2-based kinase activation. In Saccharomyces cerevisiae, which lacks the negative regulatory tail kinase Csk, wild-type Hck was more strongly activated in the presence of an SH3-binding protein (human immunodeficiency virus-1 Nef), indicating persistence of native SH3-linker interaction in an active Hck conformation. Taken together, these data support the existence of multiple active conformations of Src family kinases that may generate unique downstream signals.

The tyrosine kinases Fyn and Hck favor the recruitment of tyrosine-phosphorylated APOBEC3G into vif-defective HIV-1 particles.

The main function of Vif is to limit the antiviral activity of APOBEC3G by counteracting its packaging into HIV-1 virions. In this work, we examine the possible functional interactions between Vif, APOBEC3G, and two Src family tyrosine kinases, Fyn and Hck, present in T lymphocytes and in monocyte-macrophages, respectively. By GST pull-down, we show that the SH3 domains of Fyn and Hck, and the corresponding full-length proteins bind Vif of HIV-1. One consequence of this interaction is a reduction in their catalytic activity. Interestingly, we also observed that APOBEC3G can be phosphorylated on tyrosine in the presence of Fyn or Hck, suggesting that both kinases may regulate APOBEC3G function. Accordingly, we demonstrate that in the presence of Fyn or Hck and in the absence of Vif, the overall level of APOBEC3G incorporated into HIV-1 particles is decreased, whereas the level of encapsidation of its phosphorylated form is significantly enhanced.

Lyn regulates the cell death response to ultraviolet radiation through c-Jun N terminal kinase-dependent Fas ligand activation.

The Src-related tyrosine kinase, Lyn, plays an important role in mediating the cell cycle arrest and cell death response to genotoxic agents such as ionizing radiation. In this report we provide evidence to show that the catalytic function of Lyn is required for ultraviolet radiation (UV)- and methyl methanesulfonate (MMS)- but not for cisplatin (CDDP)- or ionizing radiation (IR)-induced cell death. Consequently, fibroblasts deficient in Lyn function were protected against cell death induction by UV and MMS, but showed normal cell death to IR and CDDP treatment. In Lyn(-/-) cells, UV-induced activation of stress-responsive kinases, Erk1/2 and p38, was normal; however, JNK activation was diminished. In addition, FasL induction by UV was also diminished in these cells. Reintroduction of wild-type Lyn restored JNK activation, FasL induction, and sensitivity to UV and MMS. A role for FasL in the cell death induction by Lyn-JNK signaling is indicated by the inhibition of cell death response by FasL neutralizing antibody. Together, the results support the presence of the Lyn-JNK signaling pathway that mediates the cell death response to UV and MMS treatment through FasL induction.

Src kinases mediate STAT growth pathways in squamous cell carcinoma of the head and neck.

Signal transducer and activator of transcription (STAT) proteins are constitutively activated in many malignancies, including squamous cell carcinoma of the head and neck (SCCHN). Previously, we reported that phosphorylation of the epidermal growth factor receptor (EGFR) is linked to activation of STATs 3 and 5 in SCCHN cells. The present study was undertaken to determine the role of Src family kinases in STAT activation and SCCHN growth. The Src family kinases c-Src, c-Yes, Fyn, and Lyn were expressed and activated by transforming growth factor-alpha stimulation in all four SCCHN cell lines examined but not in corresponding normal epithelial cells. In nine SCCHN cell lines tested, Src phosphotyrosine expression levels were highly correlated with activation levels of STATs 3 and 5. Co-immunoprecipitation analysis demonstrated interaction between c-Src and STATs 3 or 5 and EGFR in SCCHN cells, but no heterodimerization was detected between STAT3 and STAT5. SCCHN cells treated with either of two Src-specific inhibitors or transfected with a dominant-negative c-Src construct demonstrated decreased activation of STATs 3 and 5 and reduced growth rates in vitro. These results demonstrate a role for Src kinases in mediating activation of STATs 3 and 5 in concert with the EGFR in SCCHN cells. Strategies to target Src activation may contribute to the treatment of cancers that demonstrate increased levels of EGFR and STATs, including SCCHN.

Differential function of STAT5 isoforms in head and neck cancer growth control.

Up-regulation of the epidermal growth factor receptor (EGFR) is critical for the loss of growth control in a variety of human cancers, including squamous cell carcinoma of the head and neck (SCCHN). Stimulation of EGFR results in activation of mitogenic signaling pathways including Signal Transducers and Activators of Transcription (STATs). Stat5 activation has been primarily demonstrated in hematopoietic malignancies. Gene disruption studies suggest potentially distinct functions of the Stat5 isoforms, Stat5a and Stat5b, which are encoded by two genes closely linked on human chromosome 17. To determine the function of Stat5 in SCCHN growth control, we studied the expression and constitutive activation of Stat5a and Stat5b in normal and transformed human squamous epithelial cells. Increased constitutive activation of Stat5 was detected in transformed compared with normal squamous cells. Blockade of TGF-alpha or EGFR, abrogated Stat5 activation. Targeting of Stat5b using antisense oligonucleotides inhibited SCCHN growth. In addition, SCCHN cells stably transfected with dominant negative mutant Stat5b failed to proliferate in vitro. In contrast, targeting of Stat5a using either antisense or dominant negative strategies had no effect on cell growth. These results suggest that TGF-alpha/EGFR-mediated autocrine growth of transformed epithelial cells is dependent on activation of Stat5b but not Stat5a.

SH3-dependent stimulation of Src-family kinase autophosphorylation without tail release from the SH2 domain in vivo.

Src family protein-tyrosine kinase activity is suppressed by two intramolecular interactions. These involve binding of the SH2 domain to the phosphorylated C-terminal tail and association of the SH3 domain with a polyproline type II helix formed by the SH2-kinase linker. Here we show that SH3-dependent activation of the Src family member Hck by HIV-1 Nef binding or by SH2-kinase linker mutation does not affect tail tyrosine phosphorylation in fibroblasts. Surprisingly, replacement of the wild type Hck tail with a high-affinity SH2 domain-binding sequence did not affect Hck activation or downstream signaling by these SH3-dependent mechanisms, suggesting that activation through SH3 occurs without SH2-tail dissociation. These results identify SH3-linker interaction as an independent mode of Hck kinase regulation in vivo and suggest that different mechanisms of Src kinase activation may generate distinct output signals because of differences in SH2 or SH3 domain accessibility.