Phosphoproteomics of collagen receptor networks reveals SHP-2 phosphorylation downstream of wild-type DDR2 and its lung cancer mutants.

Collagen is an important extracellular matrix component that directs many fundamental cellular processes including differentiation, proliferation and motility. The signalling networks driving these processes are propagated by collagen receptors such as the ß1 integrins and the DDRs (discoidin domain receptors). To gain an insight into the molecular mechanisms of collagen receptor signalling, we have performed a quantitative analysis of the phosphorylation networks downstream of collagen activation of integrins and DDR2. Temporal analysis over seven time points identified 424 phosphorylated proteins. Distinct DDR2 tyrosine phosphorylation sites displayed unique temporal activation profiles in agreement with in vitro kinase data. Multiple clustering analysis of the phosphoproteomic data revealed several DDR2 candidate downstream signalling nodes, including SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2), NCK1 (non-catalytic region of tyrosine kinase adaptor protein 1), LYN, SHIP-2 [SH2 (Src homology 2)-domain-containing inositol phosphatase 2], PIK3C2A (phosphatidylinositol-4-phosphate 3-kinase, catalytic subunit type 2α) and PLCL2 (phospholipase C-like 2). Biochemical validation showed that SHP-2 tyrosine phosphorylation is dependent on DDR2 kinase activity. Targeted proteomic profiling of a panel of lung SCC (squamous cell carcinoma) DDR2 mutants demonstrated that SHP-2 is tyrosine-phosphorylated by the L63V and G505S mutants. In contrast, the I638F kinase domain mutant exhibited diminished DDR2 and SHP-2 tyrosine phosphorylation levels which have an inverse relationship with clonogenic potential. Taken together, the results of the present study indicate that SHP-2 is a key signalling node downstream of the DDR2 receptor which may have therapeutic implications in a subset of DDR2 mutations recently uncovered in genome-wide lung SCC sequencing screens.

Study of the PDK1/AKT signaling pathway using selective PDK1 inhibitors, HCS, and enhanced biochemical assays.

The PI3K/AKT signaling pathway has an important regulatory role in cancer cell growth and tumorigenesis. Signal transduction through this pathway requires the assembly and activation of PDK1 and AKT at the plasma membrane. On activation of the pathway, PDK1 and AKT1/2 translocate to the membrane and bind to phosphatidylinositol-(3,4,5)-trisphosphate (PIP(3)) through interaction with their pleckstrin-homology domains. A biochemical method was developed to measure the kinase activity of PDK1 and AKT1/2, utilizing nickel-chelating coated lipid vesicles as a way to mimic the membrane environment. The presence of these vesicles in the reaction buffer enhanced the specific activity of the His-tagged PDK1 (full-length, and the truncated kinase domain) and the activity of the full-length His-tagged AKT1 and AKT2 when assayed in a cascade-type reaction. This enhanced biochemical assay is also suitable for measuring the inhibition of PDK1 by several selective compounds from the carbonyl-4-amino-pyrrolopyrimidine (CAP) series. One of these inhibitors, PF-5168899, was further evaluated using a high content cell-based assay in the presence of CHO cells engineered with GFP-PDK1.

Immobilization of acetylcholinesterase in lipid membranes deposited on self-assembled monolayers.

Human red blood cell acetylcholinesterase was incorporated into planar lipid membranes deposited on alkanethiol self-assembled monolayers (SAMs) on gold substrates. Activity of the protein in the membrane was detected with a standard photometric assay and was determined to be similar to the protein in detergent solution or incorporated in lipid vesicles. Monolayer and bilayer lipid membranes were generated by fusing liposomes to hydrophobic and hydrophilic SAMs, respectively. Liposomes were formed by the injection method using the lipid dimyristoylphosphatidylcholine (DMPC). The formation of alkanethiol SAMs and lipid monolayers on SAMs was confirmed by sessile drop goniometry, ellipsometry, and electrochemical impedance spectroscopy. In this work, we report acetylcholinesterase immobilization in lipid membranes deposited on SAMs formed on the gold surface and compare its activity to enzyme in solution.

Temporal dynamics of tyrosine phosphorylation in insulin signaling.

The insulin-signaling network regulates blood glucose levels, controls metabolism, and when dysregulated, may lead to the development of type 2 diabetes. Although the role of tyrosine phosphorylation in this network is clear, only a limited number of insulin-induced tyrosine phosphorylation sites have been identified. To address this issue and establish temporal response, we have, for the first time, carried out an extensive, quantitative, mass spectrometry-based analysis of tyrosine phosphorylation in response to insulin. The study was performed with 3T3-L1 adipocytes stimulated with insulin for 0, 5, 15, and 45 min. It has resulted in the identification and relative temporal quantification of 122 tyrosine phosphorylation sites on 89 proteins. Insulin treatment caused a change of at least 1.3-fold in tyrosine phosphorylation on 89 of these sites. Among the responsive sites, 20 were previously known to be tyrosine phosphorylated with insulin treatment, including sites on the insulin receptor and insulin receptor substrate-1. The remaining 69 responsive sites have not previously been shown to be altered by insulin treatment. They were on proteins with a wide variety of functions, including components of the trafficking machinery for the insulin-responsive glucose transporter GLUT4. These results show that insulin-elicited tyrosine phosphorylation is extensive and implicate a number of hitherto unrecognized proteins in insulin action.

Adipocytes contain a novel complex similar to the tuberous sclerosis complex.

Recently we identified a novel 250 kDa protein in adipocytes that is a substrate for the insulin-activated protein kinase Akt. We refer to this protein as AS250 for Akt substrate of 250 kDa. AS250 has a predicted GTPase activating protein (GAP) domain at its carboxy terminus. This domain shows some homology to the GAP domains for Rheb at the carboxy terminus of the protein tuberin and for Rap1 in the protein Rap1 GAP. The present study further characterizes AS250. The cDNA sequence for human AS250 is reported, and the sites that undergo phosphorylation upon insulin treatment of adipocytes have been identified by tandem mass spectrometry. We have found that in adipocytes AS250 exists as a complex with a novel protein of 1484 amino acids known as KIAA1219. The complex of AS250 with KIAA1219 is notably similar to the important regulatory complex of the protein tuberin with hamartin (the tuberous sclerosis complex), in the size of its subunits, the location of the GAP domain, and its phosphorylation by Akt. In an effort to detect the cellular role of the AS250/KIAA1219 complex, we generated 3T3-L1 adipocytes that largely lack AS250 by shRNA knockdown and examined several insulin-dependent effects. The knockdown of AS250 had no effect on insulin activation of the kinases, Akt, 70 kDa S6 kinase, or ERK1/2, or on insulin-stimulated actin bundling, and it had only a slight effect on insulin-stimulated GLUT4 translocation.

Novel insulin-elicited phosphoproteins in adipocytes.

Akt is a key insulin-activated protein kinase. We searched for Akt substrates in 3T3-L1 adipocytes by means of immunoprecipitation with an Akt phosphomotif-specific antibody (PAS antibody). Four insulin-elicited phosphoproteins were isolated and identified by mass spectrometry. The identity of each protein was established by isolating the protein from lysates of untreated and insulin-treated adipocytes with an antibody specific for the protein and showing that the PAS antibody reacted only with the protein in the immunoprecipitate from insulin-treated cells. These proteins have sizes of 47, 75, 105, and 250 kDa on SDS PAGE, and have been designated pp47, 75, 105, and 250. The effect of inhibitors on the phosphorylation of the proteins, the identified sites of phosphorylation, and in vitro phosphorylation by recombinant Akt further indicated that pp47, 105, and 250 are likely to be Akt substrates, whereas pp75 may not be. pp47 and 105 are novel proteins with no known or predicted function. pp75 was previously found as a protein that associated with the colony-stimulating factor receptor, designated as Fms-interacting protein. pp250 is a novel protein with a predicted GTPase activating protein (GAP) domain for Rheb and/or Rap at its carboxy terminus. The subcellular and tissue distributions of the four proteins were determined.

Challenges and approaches for assay development of membrane and membrane-associated proteins in drug discovery.

In addition to its role as a barrier between the cytoplasm and the extracellular milieu, the cell membrane is a scaffold for a diverse collection of receptors and enzymes. The organization afforded by this scaffold serves to ensure an efficient interaction between the components of the membrane. The desire to maintain this organization in solution is a challenge for the appropriate interrogation of these biochemical components. This chapter will discuss strategies that allow biochemical analysis of membrane-associated enzymes within standard biochemical reactions. The advantages of these screening strategies in identifying valuable compounds from compound libraries and in understanding the intricacies of complex multiprotein complexes (i.e., chemotaxis) will be discussed.

Photodamage to multidrug-resistant gram-positive and gram-negative bacteria by 870 nm/930 nm light potentiates erythromycin, tetracycline and ciprofloxacin.

We have previously shown that 870 nm/930 nm wavelengths cause photodamage at physiologic temperatures in methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli via generation of endogenous radical oxygen species (ROS) and decreased plasma membrane potentials (Delta Psi p). We tested MRSA (Strain HSJ216) in vitro with sublethal 870 nm/930 nm laser energy and subinhibitory concentrations of erythromycin, tetracycline, penicillin, rifampin and trimethoprim to surmise whether photodamage could potentiate these antimicrobials. We also tested patient isolates of fluoroquinolone-resistant MRSA and E. coli with subinhibitory concentrations of ciprofloxacin. In MRSA (Strain HSJ216) we observed 97% potentiation (a 1.5 log(10) CFU decrease) with erythromycin and tetracycline. In patient isolates of E. coli, we observed 100% potentiation (>3 log(10) CFU decrease) in all irradiated samples with ciprofloxacin. To assess whether staphyloxanthin pigment conferred protection against the generated ROS, we created an isogenic carotenoid-deficient mutant of S. aureus that was significantly less tolerant of 870 nm/930 nm exposure than the wild type strain (P < 0.0001). We suggest that antibiotic potentiation results from a photobiological attenuation of ATP-dependent macromolecular synthetic pathways, similar to that observed with daptomycin, via disruption of Delta Psi p and endogenous generation of ROS. With erythromycin, tetracycline and ciprofloxacin, attenuation of energy-dependent efflux systems is also a possibility.

Near-infrared photoinactivation of bacteria and fungi at physiologic temperatures.

We examined a laser system (870 and 930 nm), employing wavelengths that have exhibited cellular photodamage properties in optical traps. In vitro, with 1.5 cm diameter flat-top projections (power density of 5.66 W cm(-2)), at physiologic temperatures, we achieved photoinactivation of Staphylococcus aureus, Escherichia coli, Candida albicans and Trichophyton rubrum. Using nonlethal dosimetry, we measured a decrease in trans-membrane potentials (DeltaPsimt and DeltaPsip) and an increase in reactive oxygen species (ROS) generation in methicillin-resistant S. aureus (MRSA), C. albicans and human embryonic kidney cells. We postulate that these multiplexed wavelengths cause an optically mediated mechano-transduction of cellular redox pathways, decreasing DeltaPsi and increasing ROS. The cellular energetics of prokaryotic and fungal pathogens, along with mammalian cells, are affected in a similar manner when treated with these multiplexed wavelengths at the power densities employed. Following live porcine thermal tolerance skin experiments, we then performed human pilot studies, examining photodamage to MRSA in the nose and fungi in onychomycosis. No observable damage to the nares or the nail matrix was observed, yet photodamage to the pathogens was achieved at physiologic temperatures. The selective aspect of this near-infrared photodamage presents the possibility for its future utilization in human cutaneous antimicrobial therapy.

The 47kDa Akt substrate associates with phosphodiesterase 3B and regulates its level in adipocytes.

We have previously described a novel putative 47kDa substrate for the protein kinase Akt (designated AS47) in 3T3-L1 adipocytes. In the present study, we have found by co-immunoprecipitation that AS47 was associated with cyclic nucleotide phosphodiesterase 3B (PDE3B) in lysates of 3T3-L1 adipocytes. The patterns of expression of AS47 and PDE3B upon 3T3-L1 adipocyte differentiation, among mouse tissues, and in adipocytes with and without the transcription factor C/EBPalpha were virtually coincident. Partial knockdown of AS47 in 3T3-L1 adipocytes with shRNA resulted in a similar reduction in PDE3B protein. These results indicate that AS47 exists in a complex with PDE3B in adipocytes and that the amount of AS47 protein regulates the amount of PDE3B.