Expression and Purification of Protein
Expression of Cdc37 (Siligardi et al., 2002
), human Hsp90α (Millson et al., 2007
) and the Hsp90-Cdc37-Cdk4 (H-C-K) and Cdc37-Cdk4 (C-K) complexes (Vaughan et al., 2006
) have been previously described. The Cdc37 S13A mutant was constructed with the QuikChange Site-Directed Mutagenesis kit (Stratagene), and the protein was expressed and purified as described for wild-type. Sf9
expressed Cdc37 was obtained as a by-product of the H-C-K complex purification. GST-PP5 was expressed in B834 cells and purified by glutathione Sepharose and gel filtration on Superdex 200 HR26/60 (GE Healthcare). PP5 protein (residues 16–499) was a generous gift of Jing Yang.
Polyclonal antibodies (Eurogentec Ltd.) were raised to a phosphopeptide corresponding to residues 9–19 of human Cdc37. Other antibodies were: polyclonal α-Cdk4 (H303) (Santa Cruz Biotechnology); monoclonal α-Cdc37 (C1) (Affinity BioReagents); PP5 (C-20), Cdc37 (H-271), C-Raf (C-20) (Santa Cruz Biotechnology); P-ERK1/2 (no. 9101, Cell Signaling); total ERK2 (a gift of Chris Marshal, ICR); and GAPDH (Chemicon). Secondary antibodies were horseradish peroxidase-linked anti-mouse and anti-rabbit IgG (GE Healthcare) and anti-goat (Abcam).
Testing Specificity of α-pSer13-Cdc37 Antibody
Cdc37, wild-type, and S13A mutant (E. coli expressed), were incubated at 30°C with 0.1 mM ATP, 1 mM MgCl2 in 25 mM Tris, and 100 mM NaCl (pH 7.5) in the absence or presence of CK2 (Promega). At each time point, samples were processed as for dephosphorylation reactions. For preparative phosphorylation, Cdc37 was further purified with a Resource Q resin (GE Healthcare) after incubation with CK2 in order to ensure a homogeneously phosphorylated sample.
Phosphatase reactions (in 50 mM Tris, 1 mM EDTA, 5 mM DTT, 2 mM MnCl2 [pH 7.5]) were stopped by boiling in SDS loading buffer for 5 min, and analyzed by SDS-PAGE and Western blot.
For dephosphorylation by λPP, Cdc37, C-K, and H-C-K with approximately equal concentrations of Cdc37 were incubated with λPP at a 1/5 (w/w) λPP:Cdc37 ratio at room temperature with a final concentration of 2 mM Na2MoO4. At specified time points, aliquots were removed and processed as described above. For gel filtration, C-K samples were incubated with/without λPP (1:10 ratio) for 1 hr at room temperature and loaded on a Superose 6 HR10/30 column (GE Healthcare) in 50 mM Tris (pH 7.5), 10 mM KCl, 10 mM MgCl2, 150 mM NaCl, 1 mM EDTA, 3 mM DTT. For H-C-K dephosphorylation and pulldowns, samples were incubated with/without λPP (10:1 ratio) at 4°C. H-C-K was buffer exchanged into 50 mM Tris (pH 7.5), 10 mM KCl, 10 mM MgCl2, and 150 mM NaCl immediately prior to setup. Phosphatase reaction buffer was modified to exclude DTT and EDTA and to include 150 mM NaCl. At specified time points, aliquots were removed and samples incubated with BSA-blocked Talon resin at 4°C for 15 min. Resin was washed with pulldown buffer (50 mM Tris [pH 7.5], 10 mM KCl, 10 mM MgCl2, 150 mM NaCl, 20 mM Na2MoO4, 0.1% NP40) and bound samples eluted by boiling with loading buffer. Proteins were quantitated by immunochemifluorescence for Cdc37 and Cdk4 (ECL plus; GE Healthcare); Coomassie staining was used for Hsp90. Data were analyzed with ImageQuant (GE Healthcare). In order to confirm H-C-K bound specifically to the resin, the experiment was repeated with a sample of H-C-K preincubated with PreScission protease (GE Healthcare) to remove the His6 tag (data not shown).
For dephosphorylation with PP5, Cdc37, C-K, and H-C-K with a [Cdc37] ~800 nM were incubated at room temperature with 35 nM PP5 and 10-fold excess of human Hsp90α, or with a C-terminal Hsp90 peptide (SRMEEVD). PP5 and activator were preincubated together for 1 hr and sample buffer exchanged into 25 mM Tris and 100 mM NaCl (pH 7.5) immediately prior to setup. Activation of PP5 by Hsp90α or the peptide was confirmed with the colorimetric substrate pNPP, as previously described (Yang et al., 2005
) (data not shown).
Coprecipitation of PP5 with Cdc37
Equal amounts of E. coli-expressed Cdc37, either unphosphorylated or phosphorylated in vitro with CK2, and preincubated with/without Hsp90α for 1 hr, were incubated with GST-PP5 or GST at 4°C for 2 hr with glutathione Sepharose resin (GE Healthcare), preequilibrated with pulldown buffer plus BSA (50 mM HEPES [pH 7.5], 150 mM NaCl, 0.1% NP40). Unbound protein was collected, the resin washed, and bound protein eluted by boiling in SDS-PAGE loading buffer. H-C-K was buffer exchanged into pulldown buffer without NP40 immediately prior to incubation with GST or GST-PP5, with or without Mg2+ (5 mM) and nucleotide (1 mM), and samples incubated overnight with glutathione Sepharose resin.
Overexpression of PP5 in Human Cancer Cells
The human colon carcinoma cell line HCT116 was cultured at 37°C, 5% CO2
in Dulbecco's modified Eagle's medium (Sigma), supplemented with MEM NEAA, 2 mM glutamine (GIBCO), and 10% FCS (PAA). PP5 open reading frame was amplified from HT29 total RNA by RT-PCR and cloned into the bicistronic eukaryotic expression vector pEFIRES-P (Hobbs et al., 1998
). HCT116 cells were transfected with Lipofectamine 2000 (Invitrogen), and cells overexpressing PP5 or the empty vector were selected by culturing with 3 μg/ml puromycin.
Cells were harvested with trypsin/EDTA and washed once in PBS. Protein samples were prepared in lysis buffer (Cell Signaling) with complete mini protease inhibitor cocktail (Roche) and quantitated by BCA protein assay (Pierce). Equal amounts of protein (25 μg) were separated by electrophoresis through 4%–20% Tris-glycine gels (Invitrogen) and transferred onto nitrocellulose. Blocking and primary antibody incubations were in casein buffer (0.5% casein, 10 mM Tris base, 150 mM NaCl, and 0.5 mM thimerosal). Specific antigen-antibody interaction was detected with a horseradish peroxidase-conjugated secondary IgG by chemiluminescence (SuperSignal West Pico chemiluminescent substrate; Pierce) and autoradiography.
Yeast Strains and Plasmids
PPT1 plus 500 bp upstream was cloned into pRS314 as EcoRI/XhoI for expression under its native promoter. PPT1 was placed under MET25 promoter control in pUT36 SpeI/XhoI. In both constructs, Ppt1 was FLAG tagged at the C terminus. K81E/R85E mutations were introduced with QuickChange site-directed mutagenesis kit (Stratagene) and verified by DNA sequencing.
The yeast strain (BY4741, ppt1Δ kanMX4
) was from EUROSCARF. The Cdc37(WT), cdc37S14A
, and cdc37S14/17E
strains were generously provided by M. Siderius and S.M. van der Vies (Hawle et al., 2007
Yeast was grown on YPD (2% [w/v] Bacto peptone, 1% yeast extract, 2% glucose or (YPGal) 2% galactose, 20 mg/L adenine). Selective growth was on dropout 2% glucose (DO) medium supplemented with appropriate amino acids. For agar growth GA sensitivity assays, overnight DO cultures (without methionine) were diluted to an optical density of 600 nm of 0.5, and ≈5 μl aliquots of a 10-fold dilution series were spotted onto DO-2% agar (without methionine) plates supplemented with the indicated level of GA. Growth was monitored over 3–5 days at 30°C.
v-Src Activation Assay In Vivo
and various cdc37
mutant alleles with or without overexpressing PPT1
were transformed with the YpRS316v-SRC (Nathan and Lindquist, 1995; Panaretou et al., 2002
). v-SRC is under control of the GAL1
promoter. Its induction and activation was analyzed as described previously (Panaretou et al., 2002
), with the exception that yeast cells were grown onto 2% glucose in order to repress GAL1
promoter. Cells were also spotted onto YPD or YPGal-2% agar. v-Src protein levels were detected with EC10 mouse antibody (Millipore) and v-Src activity with 4G10 mouse anti-pY antibody (Millipore).