All chemicals used were of the purest grade available and were obtained either from Sigma-Aldrich (Seelze), Roth (Karlsruhe) or Applichem (Darmstadt). The following cAMP-analogs were synthesised by Biolog LSI (Bremen): 8-AHA-cAMP, 8-AEA-cAMP, Sp-cAMPS, Sp-8-AHA-cAMPS, Sp-2-AHA-cAMPS, Sp-8-AEA-cAMPS, Rp-cAMPS, Rp-8-AHA-cAMPS, Rp-8-AHDAA-cAMPS. A representative scheme with synthesis and coupling of Sp-8-AEA-cAMPS to agarose beads is provided in Fig. .
Synthesis of cyclic nucleotide derivatives and coupling to agarose
All cAMP- and ω-aminoalkyl-substituted Sp-cAMPS analogs (for chemical structure see Fig. ) were synthesised as described [22
] with some minor modifications. Typically, 100 μmoles of (Sp)-8-Br-cAMP(S), (Sp)-2-Cl-cAMP(S) or (Rp)-8-Br-cAMP(S) (Biolog) and 1,000 μmoles of 1,ω-diaminoalkan (Fluka) were dissolved in 10 mL water and refluxed until no starting material was detectable by HPLC analysis (first step in the reaction pathway Fig. ). The reaction mixtures were neutralised with diluted HCl, concentrated by rotary evaporation under reduced pressure, and subsequently purified by means of semi-preparative reversed phase HPLC (YMC ODS-A 120–11, YMC). The column was washed with 100 mM NaH2
, pH 7, followed by water. Each cAMP(S) analog was eluted with a gradient from 100% water to 100% acetonitrile. The product containing fractions were collected and evaporated under reduced pressure to obtain 8-AHA-cAMP, 8-AEA-cAMP, Sp-8-AHA-cAMPS, Sp-8-AEA-cAMPS, Sp-2-AHA-cAMPS and Rp-8-AHDAA-cAMPS in yields of 60–80% with purities > 99% (by HPLC). The structure of each cAMP(S) analog was confirmed by UV/VIS spectrometry and FAB/MS or ESI/MS analysis.
cAMP(S) analogs were coupled to NHS-activated agarose beads (Affi-Gel® 10, BIO-RAD) according to the manufacturer's instructions (Fig. ). Briefly, 6.6 μmoles of cAMP(S) analog and 7.26 μmoles ethyldiisopropylamine were added per mL settled gel, suspended in DMSO. Reaction mixture was carefully shaken for 2–18 h at ambient temperature until no further consumption of starting material was detectable by analytical HPLC monitoring. Any unreacted NHS-groups of the agarose gels were blocked by addition of 20 μmoles ethanolamine per mL settled gel by incubation for 1 h. After filtration and multiple washing with subsequently 2 × 25 mL 20% ethanol, 2 × 25 mL H2O and 2 × 25 mL 30 mM NaH2PO4, pH 7, each agarose gel was stored in 30 mM NaH2PO4, 1% NaN3, pH 7 at 4°C. Ligand densities were 6 μmoles/mL 8-AHA-cAMP, 6 μmoles/mL 8-AEA-cAMP, 4 μmoles/mL Sp-8-AEA-cAMPS agarose, 5 μmoles/mL Sp-8-AHA-cAMPS agarose, 6 μmoles/mL Sp-2-AHA-cAMPS agarose and 6 μmoles/mL Rp-8-AHDAA-cAMPS agarose.
Direct binding studies of Sp-cAMPS analogs using SPR
Sp-2-AHA-cAMPS, Sp-8-AEA-cAMPS, Sp-8-AHA-cAMPS (for chemical structures see Fig. ), 8-AHA-cAMP and 8-AEA-cAMP were dissolved in 100 mM HEPES-KOH pH 8 by cautious heating (max. 70°C) and filtered. The concentrations of the stock solutions were determined via their respective extinction coefficient. CM5 sensor chip surfaces (research grade, Biacore AB) were activated for 10 min with NHS/EDC according to the manufacturer's instructions (amine coupling kit, Biacore AB). The analogs (3 mM) were injected for 7 min (running buffer: 100 mM HEPES, pH 8). Deactivation of the surface was performed with 1 M ethanolamine-HCl, pH 8.5. Each flow cell was activated, coupled and deactivated individually with a flow rate of 5 μL/min at 20°C. A reference cell (Flow cell 1) was activated and deactivated without ligand immobilisation.
All interaction analyses were performed at 20°C in 150 mM NaCl, 20 mM MOPS, pH 7 (buffer A) containing 0.005% (v/v) surfactant P20, using a Biacore 2000 instrument (Biacore AB). Binding analyses were performed by injection of 100 nM hRIα, hRIβ, hRIIα and rRIIβ (all proteins were purified classically by DEAE cellulose chromatography [48
]) at a flow rate of 10 μL/min. Association and dissociation were monitored for 5 min and 10 min, respectively. Dissociation was performed in buffer A containing 0.005% P20 in the presence or absence of 3 mM cGMP. The sensor surfaces were regenerated after each binding cycle by two short injections of 3 M guanidinium HCl. After subtracting the reference cell signal, binding data were normalised (Fig. ).
Purification of hRIα using Sp-cAMPS agaroses
Bacterial cells overexpressing R-subunit were lysed using a French Pressure Cell (Thermo Electron) in lysis buffer containing 20 mM MOPS pH 7, 100 mM NaCl, 1 mM β-mercaptoethanol, 2 mM EDTA and 2 mM EGTA (buffer A). The crude lysate was centrifuged at 27,000 g for 30 min at 4 C. Three different Sp-cAMPS agaroses (Sp-8-AHA-cAMPS, Sp-8-AEA-cAMPS and Sp-2-AHA-cAMPS agarose) were tested side by side in a one step purification strategy. 1.2 μmoles of coupled analog were used for each purification, corresponding to approximately 400 μL of agarose slurry. 12 mL supernatant from 500 mL bacterial culture were incubated with the respective affinity matrices. Binding was carried out in a batch format by gently rotating over night at 4°C. After washing the agarose seven times with 1.25 mL lysis buffer each, the protein was eluted with 1.25 mL of 10 mM cGMP in buffer B (buffer A plus 1 mM β-mercaptoethanol) by gentle rotation at 4°C for 1 h followed by an elution using 10 mM cAMP in buffer B instead of cGMP. Excess of nucleotide was removed using a PD10 gel filtration column (Amersham Pharmacia). cGMP bound to the cyclic nucleotide binding pockets was removed by extensive dialysis against buffer B.
The purification strategy of RIβ with Sp-8-AEA-cAMPS follows in principle the procedure described for RIα.
Purification of type II R-subunits using Sp-8-AEA-cAMPS agarose
The purification strategy of RII isoforms follows the procedure described for hRIα except cell lysis was performed in buffer containing 20 mM MES pH 6.5, 100 mM NaCl, 5 mM EDTA, 5 mM EGTA and 5 mM β-mercaptoethanol (buffer C) including the protease inhibitors Leupeptin (0,025 mg/100 mL, Biomol), TPCK and TLCK (each 1 mg/100 mL, Biomol). After cell lysis, the soluble protein fraction was incubated in a batch format with Sp-8-AEA-cAMPS agarose (1.4 μmol analog). The agarose was washed twice with 10 mL buffer D (20 mM MES pH 7, 1 M NaCl, 5 mM β-mercaptoethanol) and subsequently with buffer C containing protease inhibitors. Two elution steps were performed with 1 mL 25 mM cGMP in buffer C and exchanged to 20 mM MES pH 6.5, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA and 1 mM β-mercaptoethanol using a PD10 column (Amersham).
Compared purification of all R-subunits using different cAMP analog agaroses
The side by side comparison of Sp-8-AEA-cAMPS, 8-AHA-cAMP and 8-AEA-cAMP for purification was performed as described for the RI and RII purification. Crude lysate from one litre expression culture was divided into three equal aliquots and incubated in a small scale experiment with 100 μL of the respective agaroses. The cGMP elution was followed by a second elution step with 40 mM cAMP at room temperature for 30 minutes according to [18
Rp-8-AHDAA-cAMPS (for chemical structure see Fig. ) and 8-AHA-cAMP were dissolved by cautious heating (max. 60°C) in 100 mM Borate pH 8.5 with 20% DMSO and 100 mM HEPES pH 8, respectively, filtered and coupled to the sensor surface as described above. All microrecovery experiments were performed in buffer A containing 10 mM MgCl2, 1 mM ATP with 0.005% (v/v) surfactant P20 on a Biacore 3000 instrument. 250 nM PKA type I holoenzyme (R2C2), free RIα subunit or C-subunit were injected over the analog sensor surfaces in separate experiments. The association phase was monitored for 5 min followed by a 4 min washing step with running buffer. Bound protein was eluted by incubating the sensor surface with 0.2% SDS for 90 s and the eluted material was recovered in a capped vial. The sensor surfaces were regenerated by three subsequent injections of 3 M guanidinium HCl.
The recovered material from 15 repetitive cycles was pooled and applied to a 12% SDS-PAGE [49
] (Fig. ) for protein identification via MS.
Fishing of PKA holoenzyme complex with Rp-cAMPS agarose
Purified recombinant mCα, hRIα and PKA holoenzyme were each incubated with Rp-8-AHDAA-cAMPS agarose (600 pmoles coupled analog, for chemical structure see Fig. ) for 2 h at 4°C. The agarose was washed seven times with 1 mL buffer E (buffer B containing 1 mM ATP and 10 mM MgCl2). Protein was eluted with 1 mL 20 mM cAMP in buffer E by gentle rotation at room temperature for 1 h. The entire supernatant of each step was precipitated with TCA and applied to SDS-PAGE (Fig. ). For Western blotting, the samples were transferred to PVDF membrane and immunoblotted with anti-PKA C-subunit antibody (Santa Cruz Biotechnology, PKAα cat C-20) visualised by enhanced chemiluminescence (Fig. ).
Fishing of PKA holoenzyme complex with agonist and antagonist agarose from pig brain lysate
Fresh pig brain tissue was homogenised in buffer F (buffer E in the presence of protease inhibitors (complete, EDTA free, Roche), 2 mM NADH and 20 mM sucrose). After centrifugation at 13,700 g for 25 min, the supernatant was filtered and incubated with 150 μL agarose (corresponding to 1 μmole of Sp-8-AEA-cAMPS or Rp-8-AHDAA-cAMPS) over night at 4°C. The beads were washed six times with 1.5 mL buffer F. Elution was carried out with 1 mL of buffer E containing 20 mM cAMP by gentle rotation at room temperature for 1 h. All samples were precipitated with TCA for SDS-PAGE (Fig. ).
Biochemical characterisation of the proteins
The purification of the R-subunits was analysed by 12% SDS-PAGE [49
] unless otherwise noted and proteins were stained with colloidal Coomassie Brilliant Blue dye modified after Neuhoff et al.
]. After electrophoresis, remaining SDS was removed by heating and rinsing in distilled water. Overnight staining with 0.1% Coomassie®
Brilliant Blue G 250 in 5% aluminium sulphate octadecahydrate and 2% phosphoric acid resulted in intense blue bands with low background (Fig. , , , ). Protein concentration was determined by a colorimetric assay using BSA as a standard [53
]. The biological activity of the proteins was verified by a spectrophotometric phosphotransferase assay using the substrate peptide Kemptide (LRRASLG, Biosynthan) according to Cook et al.
Mass spectrometry analysis
Protein bands were excised from one-dimensional SDS-PAGE [49
] and digested in gel with trypsin according to published procedures [54
], modified by omitting all prewashing steps. After equilibrating in water, the gel pieces (Fig. , , ) were excised and homogenised via centrifugation (1,6000 g) through 10 μL pipet tips (MBP) and collected in small reaction vials (CS-Chromatographie Service). Destaining, reduction and alkylation were omitted and 25 μL digestion buffer containing 50 mM NH4
, 100 ng/μL of trypsin (sequencing grade, Promega) were added directly to the gel slurry and incubated at 50°C for a minimum 1 h [55
]. After a short centrifugation the supernatants were diluted with 40 μL of 0.3% formic acid for analysis in a nanoLC-ESI-MS/MS (nanoLC-Ultimate HPLC-system, LC Packings, Dionex coupled online to a linear ion trap mass spectrometer 4000 QTRAP™, Applied Biosystems), as described in [56
]. The MS/MS spectra were searched against a nonredundant sequence database (MSDB) using MASCOT (Matrix Science), version 1.9.05. Taxonomy was restricted to mammals with variable modifications on deamidation (NQ), myristoylation (N-Term. G), oxidation (M) and phosphorylation (ST).