The reverse in-gel kinase assay for profiling physiological kinase substrates

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1 The reverse in-gel kinase assay for profiling physiological kinase substrates Xiang Li, Bin Guan, Minu K Srivastava, Achuth Padmanabhan, Brian S Hampton & Charles J Bieberich Supplementary figures and text: Supplementary Figure 1 ERK2, Aurora A and GSK-3β substrates revealed by RIKA Supplementary Figure 2 Comparison of 2D KESTREL and 2D RIKA for fraction 12 and 13. Supplementary Figure 3 CK2 RIKA for TBB treated LNCaP lysates Supplementary Figure 4 Alignment of silver stained gel and RIKA autoradiogram. Supplementary Figure 5 Matching a silver-stained RIKA gel with a RIKA autoradiogram. Supplementary Table 1 Summary of CK2 substrates identified by Mass Spectrometry Supplementary Table 2 Summary of PKA substrates identified by Mass Spectrometry Supplementary Methods

2 Supplementary Figure 1 ERK2, Aurora A and GSK-3β substrates revealed by RIKA Supplementary Figure 1a ERK2 substrates revealed by RIKA. ERK2 Control 3 11 To reveal ERK2 substrates, a 2D ERK2 RIKA was performed using a HeLa cell nuclear extract (150 µg protein) as a source of potential substrates. Recombinant wild type rat ERK2 (upper panel) was activated in vitro by a constitutively active form of MEK1 and the activated ERK2 was polymerized in the gel at a final concentration of 25 µg/ml. A kinase activity-deficient ERK2 mutant was used as a negative control (lower panel). The arrow denotes autophosphorylation of an endogenous kinase detected in both gels.

3 Supplementary Figure 1b Aurora A substrates revealed by RIKA. Aurora Control A whole cell LNCaP extract (400 µg) was resolved on a gel containing 50 µg/ml Aurora A (upper panel). A parallel gel without kinase served as the negative control (lower panel). The arrow designates autophosphorylation of a endogenous kinase. The dashed rectangle denotes technical background that was not reproducible.

4 Supplementary Figure 1c GSK-3β substrates revealed by RIKA. MW(kDa) GSK3-β * * Control * 3 11 * A whole cell LNCaP extract (300 µg) was resolved on a gel containing constitutively active GSK3-β (25 µg/ml) (upper panel). A kinase activity-deficient form of GSK-3β was used as a negative control (lower panel). Signal designated by the red asterisks is due to activity of the endogenous kinases NM23 H1 (left) and NM23 H2 (right). The relatively low number of substrates revealed in this experiment may be due to the fact that phosphorylation by GSK- 3β generally requires priming by another kinase, for example CK1. The apparent increase in the extent of endogenous kinase activity detected in this assay compared to RIKAs with other kinases is likely due to the fact that detection of GSK-3β substrates required a longer exposure time for the RIKA gel.

5 Supplementary Figure 2 Comparison of 2D KESTREL and 2D RIKA for fraction 12 and 13. a b c d e f g

6 (a) 2D KESTREL for fraction 12 shown in Fig. 2. An aliquot from fraction 12 was incubated with (upper panel) or without (lower panel) 4 µg of CK2α as described in Methods. Spots in hatched oval, signal due to autophosphorylation of recombinant CK2α. Hatched boxes represent areas shown in Fig. 2c in the manuscript. (b) Twodimensional RIKA of fraction 12. An aliquot from fraction 12 was precipitated with TCA and focused on an 18 cm ph 4-7 IPG strip. The strip was then analyzed by RIKA with 25 µg of active (upper panel) or kinase activity-deficient (lower panel) CK2α present in the gel. Hatched boxes represent areas shown in Fig. 2d in the manuscript. (c) 2D KESTREL for fraction 13. Lysate from fraction 13 was incubated with (upper panel) or without (lower panel) 4 µg of CK2α as described in Methods. Spots in hatched oval, signal due to autophosphorylation of recombinant CK2α. (d) Two-dimensional RIKA of fraction 13. Lysate from fraction 13 was precipitated with TCA and focused on an 18 cm ph 4-7 IPG strip. The strip was then analyzed by RIKA with 25 µg of active (upper panel) or kinase activity-deficient (lower panel) CK2α present in the gel. (e) Inset in (c) (upper panel). (f) Inset in (c) (lower panel). Red arrows in (e) and (f), signal unique to the KESTREL reaction when recombinant CK2α was present. (g) Inset in (d). Red arrows designate corresponding substrates phosphorylated by recombinant CK2α in (e). Substrate 2 was not detected in the 2D RIKA gel. Multiple exposures of the 2D KESTREL autoradiogram revealed at best 15 potential CK2 substrates, while multiple exposures of the 2D RIKA radiogram revealed at least 70 CK2 substrates.

7 Supplementary Figure 3 CK2 RIKA for TBB treated LNCaP lysates a b c d e f (a-d) CK2α RIKA of a TBB-treated LNCaP extract. LNCaP cells at 80% confluence were treated with 100 µm TBB or vehicle (DMSO). 300 µgs protein from a whole-cell lysate from each treatment was analyzed in a 2D RIKA gel containing 25 µg/ml recombinant CK2α. (a) Autoradiogram of RIKA gel from the vehicle-treated LNCaP lysate. (b) Autoradiogram of a RIKA gel from the TBB-treated LNCaP lysate. (c) Inset in (a). (d) Inset in (b). Arrowheads in (d) highlight signal observed only in the RIKA using lysate from TBB-treated LNCaP cells. (e) PKA RIKA for the same untreated LNCaP lysate shown in (a), which serves as a specificity control for the TBB treatment. (f) PKA RIKA for the same TBB-treated LNCaP lysate shown in (b). Note that TBB treatment does not alter the pattern of substrate phosphorylation. Black arrows designate the activity of the endogenous kinases NM23 H1 and H2.

8 Supplementary Figure 4 Alignment of silver stained gel and RIKA autoradiogram. a b c d An LNCaP lysate ion-exchange fractionation was analyzed in a 2D RIKA gel containing 25 µg/ml of CK2α (a). An equal volume of the same lysate was applied to a 2D SDS-PAGE and silver-stained (b). Spots were excised from the silver-stained gel by aligning the gel with the autoradiogram and the identities of these spots were determined by MALDI-TOF mass-spectrometry. (c). Inset in (a). (d). Inset in (b). Arrows in (c) and (d) designate substrates matched on the gel and in the autoradiogram. Spots 1-4 were not successfully identified using MALDI. Spots 5, 6 and 7 were identified as isoforms of Nucleophosmin.

9 Supplemetary Figure 5 Matching a silver-stained RIKA gel with a RIKA autoradiogram. a b 4 7 c 4 7 d

10 An ion-exchange fraction of a whole cell LNCaP lysate was analyzed in a 2D RIKA with 1 µg/ml CK2α in the gel. The gel was silver stained (a) directly after the RIKA reaction and an autoradiogram (b) was obtained after the gel was dried. Spots were excised from the gel by matching the gel with the radiogram and the identities of these spots were determined by MALDI-TOF mass-spectrometry. (c). Inset in (a). (d). Inset in (b). Arrows in (c) and (d) designate substrates matched on the gel and in the autoradiogram. Spots 1 and 4 were not successfully identified using MALDI. Spots 2 and 3 were identified as two isoforms of the β subunit of CK2 holoenzyme, and spots 5 and 6 were identified as Ras-related protein Rab- 11A and GTP-binding protein SAR1a respectively.

11 Supplementary table 1 Summary of CK2 substrates identified by Mass Spectrometry CK2 substrates identified by LC-MS/MS Protein name Accession No. Rank Log(e) Peptides matched Sequence coverage Predicted mass (kda)/pi Known CK2 substrate? Annexin A5 P % 35.8/4.7 Yes, in the loach M. fossilis GRP94 P % 92.4/4.8 Yes HnRNP C1/C2 P % 25.2/5.0 Yes, in rat Nucleolin (C23) P % 76.4/4.6 Yes CK2 substrates excised from silver stained parallel gel and identified by MALDI-TOF Protein name Accession No. Rank Mascot score Peptides matched Sequence coverage Predicted mass (kda)/pi Known CK2 substrate? Acidic leucine-rich nuclear phosphoprotein Q % 28.8/3.9 No 32 B/ANP 32B ATP synthase subunit beta/atpb P % 56.6/5.3 No Cytosolic prostaglandin E2 synthase/pges Q % 18.7/4.4 Yes GRP94 variant 1 # Q59FC % 65.9/5.1 Yes Hepatoma-derived growth factor (HDGF) P % 26.8/4.7 No novel DNA binding/efhand/leucine zipper P % 50.3/5.1 No protein /NEFA* Nucleophosmin/B23 Q9BYG % 28.5/4.6 Yes Semenclotin * S % 40.8/9.9 No CK2 substrates excised from silver stained RIKA gel and identified by MALDI-TOF Protein name Accession No. Rank Mascot score Peptides matched Sequence coverage Predicted mass (kda)/pi Known CK2 substrate? Alpha-actinin-4 O % 104.8/5.3 No Protein disulfideisomerase A6 Q % 48/5.0 No Protein kinase C and casein kinase substrate Q9UNF % 55.7/5.1 Yes in neurons protein 2 Casein kinase II subunit beta P % 24.9/5.3 Yes 26S protease regulatory subunit 6B P % 47.3/5.1 No GTP-binding protein SAR1a Q9NR % 22.4/6.2 No Ras-related protein Rab-11A P % 24.4/6.1 No Nucleophosmin P % 32.6/4.6 Yes Summary of proteins identified by Mass Spectrometry. Top, proteins identified by LC-MS/MS from RIKA gels. In each case, CK2α was also identified and ranked either as the first or second hit. Middle, proteins identified by MALDI-TOF MS from parallel silver-stained gels. A parallel gel was run

12 together with a RIKA gel. Mascot score is -10*Log(P), where P is the probability that the observed match is a random event. Mascot scores greater than 64 are significant (p<0.05). #, This is a fragment of GRP94 including the putative CK2 sites. *, Proteins from mouse seminal vesicle extracts. The theoretical molecular weights and pis of these identified proteins are consistent with the observed molecular weight and pi on the 2D gels. Bottom, proteins identified by MALDI- TOF from silver-stained RIKA gels. Supplementary Table 2 Summary of PKA substrates identified by Mass Spectrometry PKA substrates excised from silver stained parallel gels and identified by MALDI-TOF Protein Name Accession No. Rank Mascot Score Peptides matched Sequence Coverage Predicted Mass (kda)/pi Known PKA substrate? ATP synthase subunit beta/atpb P % 56.5/5.2 No Actin, cytoplasmic 1 (Beta-actin) P % 41.7/5.3 Yes Triose Phosphate Isomerase I/ TPI I P % 26.6/6.4 No PKA substrate identified by LC-MS/MS Protein Name Accession No. Rank Score Peptides Matched Histone H2B CAB Matched Sequences R.LLLPGELAK.H R.KESYSVYVYK.V K.VLKQVHPDTGISSK.A K.AMGIMNSFVNDIFER.I Predicted Mass (kda)/pi Known PKA substrate? 13.9/10.3 Yes

13 Supplementary Methods Cell culture and TBB treatment of LNCaP cells. LNCaP cells were cultured in RPMI 1640 with 10% FBS in a humidified incubator at 37 C. Cells were treated with TBB at 100 µm (Sigma, Steinheim, Germany) or DMSO (vehicle) for 30 minutes. Plasmid construction. A clone containing the full-length human HDGF cdna was purchased from ATCC (Manassas, VA). Clones containing full-length human ANP32B, human ATPB, mouse Semenclotin, and mouse NEFA cdnas, were purchased from Open Biosystems (Huntsville, AL 35806). The open reading frame of HDGF, ANP32B and ATPB were amplified by PCR. Since the mouse Semenclotin and NEFA genes encode secretory proteins, partial sequences lacking the signal peptide were amplified by PCR. PCR products were digested with specific restriction enzymes and cloned into the 6X His-tagged protein expression vector pqe-80l (Qiagen, Valencia, CA 91355). Human PGES was amplified from LNCaP cell mrna by RT-PCR. The PCR product was cloned in a TA cloning vector (Invitrogen, Carlsbad, CA) and re-cloned into the 6X His-tagged protein expression vector prset-a (Invitrogen, Carlsbad, CA). Human wild type CK2α and kinase activity-deficient CK2α (K68M) were PCR-amplified from plasmids pzw6 and pgv15 respectively (Ref. S1, gift from D. Litchfield). PCR products were TA cloned and re-cloned into pproex-hta (Invitrogen, Carlsbad, CA). PKA (catalytic subunit α) was PCR-amplified from a clone purchased from ATCC and cloned into pqe-80l. A kinase activity-deficient PKA mutant (K72H) was derived by PCRmediated mutagenesis. Phosphatase treatment. LNCaP lysates were incubated with λ-phosphatase (New England Biolabs, Beverly, MA) at 30 C for 1 hour and precipitated with TCA. Protein IEF. Protein lysates were precipitated with TCA (Sigma, Steinheim, Germany) at a final concentration of 15%. Protein pellets were resuspended in 7 M urea, 2 M thiourea, 1% C 7 BZO (Sigma, Steinheim, Germany), 50 mm DTT, 1% IEF buffer (GE Health, Piscataway, NJ). Lysates were then applied to an immobilized ph gradient (IPG) strip and focused on an IEF cell (Bio-Rad Laboratories, Hercules, CA). Ampholytes of the same ph range were added to a final concentration of 0.5%. No additional ampholytes were added when ph 3-11 IPG strips were used. Purification of His-tagged proteins. His-tagged proteins were purified using nickel chromatography under standard conditions. In vitro kinase assays of recombinant CK2α substrates. Recombinant putative CK2α substrates purified from E. coli were dialyzed against 150 mm NaCl, 20 mm Tris, ph 7.5 prior to in vitro kinase assay. To label a recombinant protein by CK2α, approximately 5-10 µg of each recombinant protein was incubated with 0.5 µg CK2α at room temperature for 60 minutes in a 1X CK2α reaction buffer (New England Biolab, Beverly, MA) in the presence of 30 nm [γ-32p]atp and 200 µm cold ATP. The reactions were stopped by adding an equal volume of 2X Laemmli buffer. The reaction mixture was resolved by SDS-PAGE and transferred to a PVDF membrane. The PVDF

14 membrane was stained with Coommassie brilliant blue and dried prior to autoradiography. Fractionation of LNCaP lysates for RIKA. LNCaP cell lysate in 50 mm diethanolamine ph 8.8 were cleared and protein (50 mg) was applied to a 2-ml Source 15 (GE Health, Piscataway, NJ) column at 4 ºC. Proteins were eluted with a NaCl step gradient from 50 mm to 1 M. Extraction and fractionation of mouse seminal vesicle proteins. Seminal vesicles were homogenized in 50 mm Tris ph 7.5, 150 mm NaCl, 0.5% NP40 containing protease inhibitor cocktail (Roche Dignostics, Mannheim, Germany). The homogenate was centrifuged and the pellet was resuspended in 8 M urea, 20 mm glycine, ph 10.0 and homogenized again. The homogenate was centrifuged and the supernatant was mixed with 8 M urea, 20 mm, ph 10.0 to a final volume of 15 mls and applied to a 1-ml Source 15 column. The column was washed with the same buffer and eluted with a NaCl step gradient in 8 M urea. KESTREL. LNCaP lysates resuspended in 50 mm Tris, ph 7.5 were fractionated on a 1 ml Source 15 column prior to KESTREL reactions. CK2α KESTREL reactions were performed as described 7. In brief, a 15 µl aliquot of each fraction was incubated with 4 µg recombinant CK2α at room temperature for 4 minutes in the presence of 30 nm [γ- 32 P]ATP in a 30 µl reaction volume with a protease inhibitor cocktail. Reactions without recombinant CK2α were set up in parallel. All reactions were stopped with equal volume of 2X Laemmli buffer. Lysates were resolved by SDS-PAGE and subsequently transferred to PVDF membranes. For 2D KESTREL, aliquots from fractions 12 and 13 were incubated with 4 µg recombinant CK2α at room temperature for 4 minutes in the presence of 30 nm of [γ- 32 P]ATP. The reactions were stopped by precipitation with 15% TCA and prepared for 2D SDS-PAGE. Mass Spectrometry. Signal spots on RIKA gels were excised, trypsin-digested and processed on a Finnigan LCQ LC-MS/MS. MS/MS mass spectra were searched against the NCBI non-redundant protein database using the X!-tandem algorithm (thegpm.org). Spots matching signals on RIKA gels were excised, digested by trypsin, desalted by Ziptip µc18 tips, and deposited for MALDI-TOF MS peptide-mass fingerprinting using a Bruker Dalton Autoflex MS. Peptide lists were searched against SwissProt database or NCBInr database using Mascot. References. S1. Vilk, G. Saulnier, R. B. St Pierre, R.Litchfield, D. W. Inducible expression of protein kinase CK2 in mammalian cells. Evidence for functional specialization of CK2 isoforms. J Biol Chem 274, (1999).