Alexander Zaslavsky

S1P3-mediated Akt activation and cross-talk with platelet-derived growth factor receptor (PDGFR)

Akt plays a pivotal role in cell survival and tumorigenesis. We investigated the potential interaction between sphingosine‐1‐phosphate (S1P) and platelet‐derived growth factor (PDGF) in the Akt signaling pathway. Using mouse embryonic fibroblasts (MEFs) from S1P receptor knockout mice, we show here that S1P3 was required for S473 phosphorylation of Akt by S1P. In addition, S1P‐stimulated activation of Akt, but not ERK, was blocked by a PDGF receptor (PDGFR)‐specific inhibitor, AG1296, suggesting a S1P3‐mediated specific crosstalk between the Akt signaling pathways of S1P and PDGFR in MEFs. We investigated this crosstalk under different conditions and found that both Akt and ERK activation induced by S1P, but not lysophosphatidic acid (LPA), in HEY ovarian cancer cells required PDGFR but not epidermal growth factor receptor (EGFR) or insulin‐like growth factor‐I receptor (IGFR). Importantly, S1P induced a Gi‐dependent tyrosine phosphorylation of PDGFR in HEY cells. This dependence on PDGFR in S1P‐induced Akt activation was also observed in A2780, T47D, and HMEC‐1 cells (which express S1P3), but not in PC‐3 or GI‐101A cells (which do not express S1P3), further supporting that S1P3 mediates the crosstalk between S1P and PDGFR. This is the first report demonstrating a unique interaction between S1P3 and PDGFR, in addition to demonstrating a specific role for S1P3 in S1P‐induced Akt activation.

Relative hydrophobicity of organic compounds measured by partitioning in aqueous two-phase systems

Partitioning of a variety of organic compounds, the majority of which represent therapeutic drugs, was examined in an aqueous dextran-polyethylene glycol (Dex-PEG) two-phase system containing 0.15 M NaCl in 0.01 M sodium phosphate buffer at pH 7.3 and in an octanol-buffer (0.15 M NaCl in 0.01 M sodium phosphate buffer, pH 7.3) system. The possibility of introducing compounds to be partitioned in an aqueous two-phase system with dimethyl sulfoxide, and the effect of this solvent on the solute partitioning was explored. Relative hydrophobicity of the compounds was estimated and expressed in equivalent numbers of methylene units. Comparison of the results obtained for several subsets of compounds in the octanol-buffer and in aqueous Dex-PEG two-phase systems clearly demonstrates the advantage of aqueous two-phase partitioning for the hydrophobicity measurements over partitioning in octanol-buffer system.

High throughput characterization of structural differences between closely related proteins in solution.

Partitioning of a protein in an aqueous two-phase system (ATPS) is governed by interactions of the protein with aqueous media in the two phases. Here we describe how partitioning of proteins in a set of ATPS of different compositions can be used to quantify differences between 3D structures of closely related proteins. We also provide perspective on practical applications of the technology when comparative analysis of the higher-order structure of proteins is desired.

Peptides partitioning in an aqueous dextran-polyethylene glycol two-phase system

Partitioning of glycine, lysine and aspartic acid and their oligopeptides in an aqueous dextran-polyethylene glycol two-phase system containing 0.15 M NaCl in 0.01 sodium phosphate buffer, pH 7.3 and 0.11 M sodium phosphate buffer, pH 7.3 was examined. Relative hydrophobicity of the amino acid residues and peptide bonds was estimated and expressed in equivalent numbers of methylene units. Analysis of a series of reversed di- and tripeptides in terms of relative hydrophobicity showed that the additivity principle does hold for the hydrophobicity of short peptides. The relative hydrophobicity of peptides is affected by the ionic composition of aqueous media as well as by the type of amino acid residues forming peptide bonds in a given peptide sequence.

Sphingosine-1-phosphate induces a PDGFR-dependent cell detachment via inhibiting β1 integrin in HEK293 cells

Several different types of interactions between sphingosine‐1‐phosphate (S1P) receptors and platelet‐derived growth factor receptor (PDGFR) have been revealed recently. In this work, we used HEK293 cells to further investigate the potential crosstalk. Interestingly, we observed that S1P specifically induced a PDGFR‐dependent cell detachment in HEK293 cells, which could be inhibited by AG1296, a specific inhibitor for PDGFR. EGFR on the other hand, did not have any effect on cell detachment. The detachment was extracellular matrix (ECM) protein specific, suggesting the involvement of specific integrin molecules. When β1 integrin was engaged into an active state, S1P‐induced cell detachment was blocked, suggesting that S1P induced an inside‐out inhibitory effect on β1 integrin. Gi protein and ERK activation were required for the cell detachment induced by S1P, suggesting an endogenous receptor for S1P is likely to be involved.