Supplemental Experimental Procedures
Reagents
pGEX2T-CrkII (human) was gifted by Dr. Michiyuki Matsuda. pSLX-Abl 1b (human) was
gifted by Dr. Giulio Superti-Furga. Plasmids for human Crk II (pCAGGS) have been described
previously. pGEX2T-CrkII (human) was provided by Dr. Michiyuki Matsuda. Plasmids
encoding Flag tagged Fc, Fc-Tyro3 and Fc-Mer (mouse) were provided by Dr. Sergei Kotenko.
The following constructs were generated: human Crk II SH3C (230-304) and full length human
Crk II in pET42a with a Tev protease cleavage site in between the polyHis tag and the ORF, Crk
II and EYFP- Crk II (human) in pMSCV. Her1, Her2 and Her3 were provided by Paul Van
Bergen. pLKO.1 Crk shRNA was purchased from Sigma. pLKO.1 scrambled shRNA, psPAX2
and pMD2.G were purchased from Addgene. Mutants Y221F, Y239F, Y251F, Y221F/Y239F
and Y221F/Y251F were
generated by using the QuickChange site-directed mutagenesis kit
(Stratagene). Anti-pY221 Crk, anti-actin, anti-Csk and anti-pY245 Abl were purchased from Cell
Signaling Technology, anti-Abl (Ab-3) from Calbiochem, anti-CrkII, anti-Flag and glutathioneagarose beads from Sigma, anti-pY (PY99) and Protein G –agarose beads from Santa Cruz
Biotechnology, HRP-conjugated goat anti-mouse and goat anti-rabbit from Jackson
Immunologicals, Nickel-NTA beads from Qiagen, Millex transwell inserts from Millipore, Type
I Collagen from BD. Transfections were performed with the Xtremegene HP reagent according
to the manufacturers’ protocol (Roche). Human EGF was purchased from Life Technologies,
human HGF, human Heregulin-beta1 and murine PDGF-BB were purchased from Peprotech.
Phosphopeptides pY239 and pY251 with sequences Biotin-LC-KLQNGPIpYARVIQKRV and
Biotin-LC-KRVPNApYDKTALALE respectively, were purchased from Anaspec Inc.
Cell culture
HEK 293T, MDA-MB-468, MDA-MB-231 and NIH 3T3 cells were purchased from ATCC.
HEK 293T, MDA-MB-468 and MDA-MB-231 were maintained in DMEM with 10% Fetal
Bovine serum while NIH 3T3 cells were maintained in DMEM with 10% Calf Serum. Crk (-/-)
MEFs were provided by Tom Curran.
Protein preparation
Isotopically labeled samples for NMR studies were prepared by growing bacterial cells in M9
minimal media supplemented with 1 g/l of
15
NH4Cl and 2 g/l of
13
C6-glucose at 37 C. Protein
synthesis was induced by addition of 0.5 mM IPTG at OD600 = 0.4. The cytosolic fraction and
membrane fractions were separated by centrifugation at 70,000g. The lysate was loaded on NiNTA agarose resin (GE) pre-equilibrated with Tris buffer and 1M NaCl. Finally, the sample was
concentrated and applied to a Superdex 200 size-exclusion column (GE). For ITC, protein
samples were dialyzed against ITC buffer containing 50 mM KPi (pH 6.8), 150 mM NaCl, and 1
mM TCEP.
GST-pull down experiments
GST-fusion proteins (bait) were bound to a glutathione-agarose column, washed twice with 50
mM Tris, 150 mM NaCl, 0.1% Triton-X-100, pH 7.5. Lysates of MDA-MB-468 cells prepared
in HNTG buffer (1% Triton-X-100) were added to columns bound to the bait. The column was
washed again twice and then proteins bound to the bait were eluted with 10mM Glutathione in
50 mM Tris, pH 8.0. Samples were analyzed by SDS PAGE followed by in gel staining with
SYPRO RUBY. Specific bands or entire lanes were cut out and analyzed by LC-MS/MS on an
Orbitrap instrument.
Supplemental Figure Legends
S1A, MDA-MB-468 cells were transfected with the plasmids indicated and lysates were analyzed
by western blotting with the antibodies shown on the left.
S1B, Quantification of western blots from Fig 2 by densitometry using ImageJ. Threshold was
applied to western blots which were grouped by the antibody used within each figure. Left, In
cases where a peak was not obtained after setting the threshold, arbitrary values were set for
plotting bar graphs as follows: value was set to 0.1 for all three lanes of a blot in case no peaks
were obtained in the control and induced samples. Value was set to 10 for induced samples in
case no peak was obtained in the control but peaks were obtained in the induced samples. Right,
Band intensity of pY221, pY239 and pY251 blots normalized to total Crk levels from Fig 2C
(lanes 7 and 8). The quantification for fig 2A and panel 4 in fig 2B along with raw values and all
other calculations are shown in supplemental table T5.
S2, Overlay of 1H-15N HSQC of Crk (blue) and pCrk (red). No drastic changes were observed in
the residues lining the SH3N-PPII binding pocket – F144, W169 and Y186.
S3A, Composite figure of normalized cell index of EYFP, EYFP-hCrk and EYFP-hCrk Y251F
expressing MDA-MB-468 cells from independent experiments to record motility towards EGF
as shown in Fig 5D (middle panel). ‘**’ indicates p<0.05.
S3B, 293T cells were transfected with the plasmids indicated and lysates were analyzed by
western blotting with the antibodies shown on the left. The Crk SH3N-linker-SH3C unit (123304) is referred to as S-S.
S4, MS/MS spectrum of a doubly-charged ion (m/z 574.80) corresponding to the peptide
sequence of
180GMIPVPYVEK189.
The observed y- and b-ion series confirmed the peptide
sequence. No peptides containing the phosphorylated Y186 residue were observed.
S5, Multiple sequence alignment of CrkII and CrkL from the species indicated on the right.
Yellow indicates conserved regions, pink and green indicate progressively diverged regions.
Supplemental Tables
ST1, Raw values from quantification of the SH2 – rosette assay for binding of GST-SH2
domains to pY239 and pY251 Crk phosphopeptides. Values are in arbitrary units. Selectivity
index calculations are shown.
ST2, Bio-informatic analyses of SH2 binding partners for the pY239 and pY251 motifs using
NetPhorest. Selectivity index calculations are shown.
ST3, Table of proteins identified by LC-MS/MS from pull downs with phosphorylated GST,
GST- SH3C (unphosphorylated) and phosphorylated GST-SH3C. The bands indicated in Fig 4G
were analyzed. Csk had the highest protein sequence coverage of 31% in the phosphorylated
GST-SH3C sample and 0% in the unphosphorylated GST-SH3C sample.
ST4, Table of proteins identified by LC-MS/MS from pull downs with GST-pSH3C WT,
Y239F, Y251F and Y239F/Y251F. Portion of gel above the bait in each lane was analyzed in Fig
4H.
ST5, Quantification of western blots from figure 2.