Activation of Erk after expression of human SWAP70 in NIH3T3 cells and its effect on the growth of the cells.

<p>A, Activation of MEK was examined by phosphorylation of Erk, p42, and p44. The assay was done by Western blotting with anti-Erk or anti-phospho-Erk. B, 1×10⁵ cells were plated onto 6-cm dishes, and cell numbers were counted every day. Medium was changed every other day. The dotted and broken lines show growth curves when 10 µM U0126 and 10 µM PD98059 were added to the medium, respectively.</p

Expression of mutant SWAP70 s and morphology of the transfectants.

<p>A, Expression of FLAG-tagged SWAP-70 was detected by Western blotting with an anti-FLAG antibody. B, Morphology of the transfectants. The cells expressing mutant SWAP-70 s were slightly shorter than the parental or the wild-type SWAP-70-expressing cells.</p

Growth of cells expressing mutant SWAP-70 under low serum conditions.

<p>2×10⁵ cells were plated onto 6-cm dishes in the medium containing 1% calf serum, and cell numbers were counted every other day. The medium was exchanged every other day. For reference, the growth curves of the parental cells are shown in every graph. The symbols for the cell lines are shown in the right-hand boxes.</p

Effect of nutrient starvation on the cells expressing mutant SWAP-70 s.

<p>1×10⁶ cells were plated onto 6-cm plates, and cell number was counted every day. Medium was not changed during the experiments. For reference, the growth curves of the parental cells are shown in every graph.</p

Soft agar colony formation of the cells expressing mutant SWAP-70 s.

<p>(A) 1×10⁴ cells were plated into soft agar in 6-well plates. Cells were observed for 10 days. On the sixth day, agar overlay was performed. (B) Similar experiments were performed in the presence of 10 µM PD98059.</p

SWAP-70 gene and the mutations found in human tumors.

<p>The SWAP-70 coding region is shown in the middle. It has an EF-hand-like domain, which contributes to binding of activated Rac1. In the center portion, the protein has a PH domain that is responsible for PIP₃ binding activity. This domain is followed by a long coiled-coil domain, which may contribute to the formation of dimers or trimers. At the very-carboxyl terminal end, it has an F-actin binding domain. This region may be masked in the normal conformation; but upon activation, it may become exposed. The arrows show the positions of mutations. The amino acid changes are shown by single letters. The organs are also shown.</p

Growth curve of the cells expressing mutant SWAP-70 s.

<p>1×10⁵ cells were plated onto 6-cm dishes, and cell numbers were counted every day. The medium was changed every other day. For reference, the growth curves of the parental cells are shown in every graph. The symbols for the cell lines are shown in the right-hand boxes.</p

Comprehensive Binary Interaction Mapping of SH2 Domains via Fluorescence Polarization Reveals Novel Functional Diversification of ErbB Receptors

<div><p>First-generation interaction maps of Src homology 2 (SH2) domains with receptor tyrosine kinase (RTK) phosphosites have previously been generated using protein microarray (PM) technologies. Here, we developed a large-scale fluorescence polarization (FP) methodology that was able to characterize interactions between SH2 domains and ErbB receptor phosphosites with higher fidelity and sensitivity than was previously achieved with PMs. We used the FP assay to query the interaction of synthetic phosphopeptides corresponding to 89 ErbB receptor intracellular tyrosine sites against 93 human SH2 domains and 2 phosphotyrosine binding (PTB) domains. From 358,944 polarization measurements, the affinities for 1,405 unique biological interactions were determined, 83% of which are novel. In contrast to data from previous reports, our analyses suggested that ErbB2 was not more promiscuous than the other ErbB receptors. Our results showed that each receptor displays unique preferences in the affinity and location of recruited SH2 domains that may contribute to differences in downstream signaling potential. ErbB1 was enriched versus the other receptors for recruitment of domains from RAS GEFs whereas ErbB2 was enriched for recruitment of domains from tyrosine and phosphatidyl inositol phosphatases. ErbB3, the kinase inactive ErbB receptor family member, was predictably enriched for recruitment of domains from phosphatidyl inositol kinases and surprisingly, was enriched for recruitment of domains from tyrosine kinases, cytoskeletal regulatory proteins, and RHO GEFs but depleted for recruitment of domains from phosphatidyl inositol phosphatases. Many novel interactions were also observed with phosphopeptides corresponding to ErbB receptor tyrosines not previously reported to be phosphorylated by mass spectrometry, suggesting the existence of many biologically relevant RTK sites that may be phosphorylated but below the detection threshold of standard mass spectrometry procedures. This dataset represents a rich source of testable hypotheses regarding the biological mechanisms of ErbB receptors.</p> </div

Competitive inhibition binding curves of protein-peptide interactions detected by FP.

<p>Nine ErbB phosphotyrosine sites were queried against five proteins: (<b>A</b>) RASA1-N, (<b>B</b>) SRC, (<b>C</b>) GRB7, (<b>D</b>) GRB2, and (<b>E</b>) PTK6. The predicted binding affinities of competitor peptide curves are color-coded as follows: red (<i>K_D<</i>1), purple (1≤ <i>K_D<</i>5), blue (5≤ <i>K_D<</i>20), and black (<i>K_D</i> ≥20). “OP” refers to the original rhodamine-labeled peptides and “CP” to the unlabeled competitor peptides, which have been numbered in the figure with sequences.</p

Comprehensive SH2 domain recruitment potential of the ErbB family as determined by high-throughput fluorescence polarization (HT-FP).

<p>Color-coded heat maps represent apparent dissociation constants (K_Ds) for FP interactions between SH2/PTB domains and phosphopeptides representing all potential ErbB1, ErbB2, ErbB3, and ErbB4 phosphotyrosine sites; black boxes indicate interactions that are too weak to be detected by the assay. Homologous ErbB peptides with identical amino acid residues from +1 to the +4 position relative to the phosphotyrosine (X) are indicated with an asterisk followed by the number (in order of occurrence) of the homologous receptor. Sequences of peptides used are indicated for each homologous receptor site, in which a small “d” denotes the pre-charged aspartic acid (Asp) residue on the peptide synthesis resin and not a naturally occurring Asp. NS refers to peptides that were unable to be synthesized, while NI refers to synthesized peptides that produced no positive hits in the study; therefore we cannot confirm nor deny interactions at these sites with our assay. Rows of the heatmaps for these peptides are grayed out to indicate that our FP assay could neither confirm nor deny positive or negative interactions from these peptides.</p