Jul 30, 1987 - Eric A. First$ and Susan S. Taylor8. From the Department of ... Treatment of the C-subunit with each reagent led to the stoichiometric ...... Takio, K., Smith, S. B., Krebs, E. G., Walsh, K. A., and Titani,. K. (1984) Binchemistry 23 ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 263, No. 11, Issue of April 15,pp. 5170-5175,1988 Printed in U.S.A.
Subunit InteractionSites between the Regulatory and Catalytic Subunits of CAMP-dependentProtein Kinase HETEROBIFUNCTIONAL CROSS-LINKING REAGENTS LEAD TO PHOTODEPENDENT AND PHOTOINDEPENDENT CROSS-LINKING* (Received for publication,July 30, 1987)
Eric A. First$ and Susan S. Taylor8 From the Department of Chemistv, University of California, San Diego, La Jolkz, California92093
Heterobifunctionalcross-linkingreagents have been Several approaches have allowedus todeduce a few regions introduced into the catalytic subunit of CAMP-depend- in both subunits that must come in close contact when the ent protein kinase as potential probes for identifying two subunits aggregate. These regions have been targeted as specific points of contact between the catalytic (C)- sites where heterobifunctional cross-linking reagents might subunit and thetype 11 regulatory (R") subunit in the be introduced in order to identify broader areas of subunit holoenzyme complex. Since at least one of the 2 cys- contact. For example, the type I1 form of the R-subunit is teine residues in the C-subunitis known to be in close characteristically autophosphorylated by the C-subunit, and proximity to the interaction site between the C-subunit Rangel-Aldeo and Rosen (2) established that this autophosand the R1'-subunit, these cysteines were chosen ini- phorylation isan intramolecular event. Sequences of the tially as targets for covalent modification by two heterobifunctional cross-linking reagents, p-azidophena- phosphorylation sites in substrates of CAMP-dependent proN-4-(azidophenylthio)phthalimide. tein kinase have revealed that all possess two basic residues cy1bromideand Treatment of the C-subunit with each reagent led to on the amino-terminal side of the phosphorylated serine (3). the stoichiometric modification of Cys-199 and Cys- The R"-subunit has the sequence Asp-Arg-Arg-Val-Ser-Val343. In each case, themodifiedC-subunit was still Cys-Ala, and the serine residue contained in this sequence, capable of forming a stable complexwith the R"-sub- Ser-95, is phosphorylated readily by the C-subunit either in unit. Both modified C-subunits also could be covalently the presence or absence of cAMP (4). This evidence strongly suggests that the R"-subunit inhibits the catalytic activity of cross-linked to the R"-subunit; however, the mechathe C-subunit, at least in part, by competing with substrates nismsfor cross-linking differed. Catalyticsubunit modifiedby p-azidophenacyl bromide was cross-linked for the active site. In addition, phosphorylation of Ser-95 decreases the affinity of the R"-subunit for the C-subunit by to the R"-subunit in a photodependent manner by a mechanism that was maximal when holoenzyme was a factor of 10 (5), supporting the hypothesis that this autoformed and CAMP was absent. In contrast, the C-sub- phosphorylation site is a specific point of contact between the unitmodifiedby N-4-(azidophenylthio)phthalimide two subunits. At least three isoforms of the R-subunit have was cross-linked totheR"-subunitby a mechanism been identified thus far (6-4,and each has conserved these that was independent of photolysis.In this case, cross- 2 arginine residues although in some cases the autophospholinking was enhanced by the presence of CAMP. This rylation site has been lost. cross-linking was the result ofa disulfide interchange In theC-subunit, the active site region has been identified between a modified cysteine in the C-subunit and an by labeling with the ATP analogue, p-fluorosulfonylbenzoyl unmodified cysteine in the R"-subunit. adenosine (FSBA)' (9, 10). This reagent contains a reactive sulfonyl group at a location that would be in close proximity to the y-phosphate of ATP and was found to react with LysThe catalytic activity of CAMP-dependent protein kinase 72 of the C-subunit. If Lys-72 is close to the y-phosphate of (EC 2.7.1.37) is regulated by the interaction between the MgATP, presumably it also is in close proximity to residues catalytic (C) subunits and the regulatory (R) subunits that that interact with the autophosphorylation site of the R"make up the holoenzyme form of the enzyme. In the holoen- subunit. This is consistent with the observation that, when zyme, the C-subunitandR-subunit aggregate to form an the C-subunit is part of the holoenzyme complex, Lys-72 is inactive %C, tetramer. Binding of cAMP decreases the affin- protected against modification by FSBA. Alkylation of the ity of the R-subunit for the C-subunit byfive orders of cysteines in the C-subunit also inactivates the free C-subunit magnitude (l),suggesting that at least two distinct confor- and, in the absence of CAMP,the R"-subunit protects the Cmational states exist for the R-subunit, one having a high subunit from this inactivation (11,12). Analysis of the modaffinity for cAMP and the otherhaving a high affinity for the ification of holoenzyme by iodoacetic acidin thepresence and absence of cAMP indicated that modification of Cys-199 was C-subunit. ~~
* This work was funded by United States Public Health Service Grant GM19301 (to S. S. T.)-The costs of publication of this article were defrayed in part by the payment of page charges. This article must thereforebe hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Supported in part by United States Public Health Service Training Grant AM07233. Present address: Dept. of Chemistry, Imperial College, London SW7 2AY, England. § To whom reprint requests should be addressed.
~~
' The
abbreviationsused are: FSBA, p-fluorosulfonylbenzoyladenosine; APTP,N-4-(azidophenylthio)phthalimide;C-subunit, catalytic subunit; CH,CN, acetonotrile; HEPES, N-2-hydroxyethylpiperazineN'-2-ethanesulfonic acid IAA, iodoacetic acid NEM, N-ethylmaleimide; PAPB, p-azidophenacyl bromide; R"-subunit, type I1 regulatory subunit; R1Iu,type I1 regulatory subunit treated with urea to remove bound cyclic nucleotide; SDS, sodium dodecyl sulfate; HPLC, high performance liquid chromatography; DTNB, 5,5'-dithiobis-(2nitrobenzoic acid).
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CAMP-dependent Protein Kinase:Cross-linking R- and C-Subunits responsible for the inactivation of the C-subunit (12). Since this residue is selectively protected from alkylation in the holoenzyme but not in the dissociated subunit, Cys-199 is a second residue on the C-subunit that maycome in close contact with the R"-subunit. Treatment of the C-subunit with a peptide analog, which contained a cysteine disulfide bonded to thionitropyridine at the position where the phosphorylatable serine typically wouldbe located, inactivated the Csubunit by forming a disulfide bond with Cys-199 (13). This is further evidence that Cys-199 is in close proximity to the peptide recognition site of the C-subunit. In order to look more extensively at specific interaction sites between the R"-subunit and the C-subunit, covalent derivatives of the C-subunit were prepared which introduced heterobifunctional groups. Since at least 1cysteine is thought to be in close proximity to the interaction site between the R"-subunit and the C-subunit, reagents were selected that specifically targeted sulfhydryl groups. The two reagents that were used were p-azidophenacyl bromide (PAPB) and N-4(azidopheny1thio)phthalimide (APTP). Both of these reagents contain a photoactivatable azide as well as anelectrophilic group that will target sulfhydryl residues. Inother systems, PAPB hasbeen shown to form anoncleavable crosslink (14). In contrast, cross-links formed with APTP can be cleaved in thepresence of reagents that reduce disulfide bonds (15). Both reagents have the advantage that the modified Csubunitcan be reassociated with the R"-subunit to form holoenzyme prior to activation of the photosensitive azide. EXPERIMENTAL PROCEDURES
Materials-Chemicals and reagents were obtained from the following sources: PAPB and APTP(Pierce Chemical Co.); synthetic peptide, Leu-Ala-Ala-Thr-Ser-Leu-Gly (Peptide-Oligonucleotide Facility, University of California, San Diego); ["C]iodoacetic acid (IAA) and t3*P]ATP(Amersham Corp.); 5,5'-dithiobis-(2-nitrobenzoicacid) (DTNB) (Sigma); N-ethylmaleimide (NEM) (Behring Diagnostics); and N"-p-tosyl-L-phenylalaninechloromethyl ketone HC1-treated trypsin (United States Biochemical Corp.). Protein Purification-The R"-subunit andthe C-subunit were purified from porcine heart as described by Nelson and Taylor (16). Typically, 8 kg of heart yielded 70-90 mg of the C-subunit and 30-50 mg of the R"-subunit. The C-subunit was stored frozen in 40 p~ potassium phosphate (pH 6.5) containing 2 pM EDTA, 5 p~ 2mercaptoethanol, and 10% glycerol (buffer A). The R"-subunit was eluted from CAMP-affinity resin with 25 PM CAMP, concentratedto approximately 1 mg/ml using an Amicon ultrafiltration apparatus, and stored frozen in 25 ptM potassium phosphate (pH 6.5) containing 25 p~ CAMP, 2 p~ EDTA, and 5 p~ 2-mercaptoethanol (buffer B). Cyclic nucleotide-free R"-subunit was prepared by urea treatment as described by Builder et al. (17) and isdesignated as R". Alternatively Rn-subunit could be prepared by exhaustive dialysis (2-3 days) of R"(cGMP)* against 8 1-liter changes of 10 p~ potassium phosphate (pH 6.5), 2 *M EDTA, 2 M NaCl, and 5 FM 2-mercaptoethanol. Type I1 holoenzyme was prepared by combining the R"-subunit with a 10% excess of the C-subunit followed by overnight dialysis at 4 "C against 10 mM potassium phosphate (pH 6.5) containing 2 mM EDTA and 10% glycerol (buffer C). Free C-subunit that remained after formation of holoenzyme was removed by incubation with CL CM-Sepharose (0.5ml of resin/mg of free C-subunit). Enzyme activity was monitored using the synthetic peptide, LeuArg-Arg-Trp-Ser-Leu-Gly, according to the coupled assay described by Cook et al. (19). Modification of the C-subunit and the R"-subunit-The C-subunit (1 mg/ml) was dialyzed against buffer C at 4 "C to remove excess 2mercaptoethanol. The C-subunit was then made 100 mM in HEPES, pH 7.4, and 1 mM in p-azidophenacyl bromide (PAPB) using a 10 mM stock solution of PAPB inmethanol. The reaction was incubated at room temperature in the dark, and, on completion of the reaction, excess reagents were removed by dialysis against buffer C at 4 "C. The C-subunit modified by APTP was formed by incubating Csubunit with 100 mM HEPES, pH 7.4, and 0.5 mM APTP using a stock solution containing 10 mM APTP in acetonitrile. Incubation
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was carried out a t 20 "C, and excess reagents were removed bydialysis against buffer C. These same procedures also were used for the modification of the R"-subunit by APTP as well as for the modification of both the R"-subunit and theC-subunit by NEM. The extent of modification of the C-subunit was followed by loss of activity. In addition, aliquots of both the C-subunit modified by PAPB and theC-subunit modified by APTP were labeled with ["C] IAA (100nCi/ml, 50pCi/mmol) as describedpreviously (16) and then digested with Na-p-tosyl-L-phenylalanine chloromethyl ketone HCItreated trypsin (1/50,w/w) overnight at 37 "C. The resulting tryptic peptides were resolved by high performance liquid chromatography (HPLC) on a Vydac C-18 reverse phase column using a flow rate of 1 ml/min and a gradient from 0 to 40% B in 120 min where the A solvent was 0.1% trifluoroacetic acid, and theB solvent was CHsCN. Formation of Holoenzyme-The integrity of holoenzyme formed from the modified C-subunits was assessed by ion-exchange chromatography. Free C-subunit has a PI greater than 7.5 and at pH6.5 in low salt (
