Phosphorylase Kinase, a Metal Ion-dependent Dual Specificity Kinase*

Phosphorylase kinase is shown to be a dual specificity kinase. The specificity of phosphorylation is determined by divalent cation. Mg2+ causes seryl phosphorylation of phosphorylase b, but Mn2+ activates tyrosine phosphorylation of angiotensin 11. In contrast to seryl phospho- rylation, the tyrosine kinase activity of holoenzyme is not regulated by Ca2+. Preincubation of the holoenzyme with Ca2+, Mg2+, and ATP that causes autophosphorylation activates tyrosine kinase activity. The tyrosyl ki- nase activity is a property of the y subunit. Addition of varying amounts of Mn2+ to a truncated form of the y subunit of phosphorylase kinase containing MgATP inhibits serine kinase but activates tyrosine kinase activity. This result along with an oxidative reaction caused by Cu2+ and site-directed mutagenesis of the putative catalytic base inhibiting both serine and tyrosine kinase activity suggest that one active site is involved in both activities. Kinetic studies with Mn2+ and ATP show that IC, for nucleotide is not changed with a seryl or tyrosyl substrate. The V, values are different, and the value for tyrosyl phospho~lation is similar to other tyrosyl ki- nases. We propose two conformations for the active site; one favors seryl phosphorylation, and the second tyrosyl phosphorylation

Phosphorylase kinase is shown to be a dual specificity kinase. The specificity of phosphorylation is determined by divalent cation. Mg2+ causes seryl phosphorylation of phosphorylase b, but Mn2+ activates tyrosine phosphorylation of angiotensin 11. In contrast to seryl phosphorylation, the tyrosine kinase activity of holoenzyme is not regulated by Ca2+. Preincubation of the holoenzyme with Ca2+, Mg2+, and ATP that causes autophosphorylation activates tyrosine kinase activity. The tyrosyl kinase activity is a property of the y subunit. Addition of varying amounts of Mn2+ to a truncated form of the y subunit of phosphorylase kinase containing MgATP inhibits serine kinase but activates tyrosine kinase activity. This result along with an oxidative reaction caused by Cu2+ and site-directed mutagenesis of the putative catalytic base inhibiting both serine and tyrosine kinase activity suggest that one active site is involved in both activities. Kinetic studies with Mn2+ and ATP show that I C , for nucleotide is not changed with a seryl or tyrosyl substrate. The V, values are different, and the value for tyrosyl phospho~lation is similar to other tyrosyl kinases. We propose two conformations for the active site; one favors seryl phosphorylation, and the second tyrosyl phosphorylation is caused by the binding of divalent cation at a second metal ion binding site.
Protein kinases modulate diverse physiologic processes by catalyzing the incorporation of a phosphate group into the side chain of various amino acid residues of proteins. Several amino acids can serve as acceptors of phosphate, but the alcoholic group of serine and threonine and the phenolic group of tyrosine are the major targets for protein kinases (1,2). Recently, it has been shown that the imidazole group of histidine can be phosphorylated by histidine protein kinase (3). Most protein kinases show selectivity and phosphorylate the side chain of only one of the functional groups, but some protein kinases have been found to phosphorylate more than one type of amino acid residues and are termed dual specificity kinases (4). M A P 1 kinase kinase, a member of this family, can autophosphorylate and phosphorylate MAP kinase on alcoholic and phenolic * This research was supported by Grant GM-09587 from the National Institutes of Health. This is Journal Paper 5-15379 of the Iowa Agrculture and Home Economics Experiment Station, Ames, IA, Project 2120. The costs of publication of this article were defrayed in part by the u a~u e~~s e m e n~" in accordance with 18 U.S.C. Section 1734 solely to payment of page charges. This article must therefore be hereby marked indicate this fact. $ To whom all correspondence should be addressed.
The abbreviations used are: MAP, mitogen-activated protein; groups (5-7). These dual specificity kinases are not very different in primary structure of their catalytic cores from that of the tyrosine kinases and the serinelthreonine kinases (4). Little is known about what structural features and factors are import a n t for dual specificity.
Protein kinases require a divalent cation along with a nucleoside triphosphate for effective phosphorylation. Serine/ threonine kinases use Mg2+ more effectively than Mn2+, but tyrosyl kinases differ because Mn2+ stimulates phosphorylation equal to or better than that caused by Mg2+ (8). Several serine/ threonine protein kinases, including CAMP-dependent protein kinase (9) and phosphorylase kinase (10,11), have been shown to bind more than one metal ion. The binding of the additional metal ion may either activate or inhibit kinase activity. Results obtained from x-ray crystallography of the catalytic subunit of CAMP-dependent protein kinase (12,13), a serine kinase, show that two metal ion binding sites exist and these sites are in close proximity. Occupancy of the first site activates phosphotransfer activity, but binding at the second site inhibits the reaction. Phosphorylase kinase, a calcium-dependent protein kinase that regulates glycogen metabolism (14,151 by phosphorylating a single seryl residue per monomeric unit of phosphorylase b (841 amino acid residues), also contains two metal ion binding sites associated with its catalytic subunit. In this instance, binding of divalent cation, i.e. Mg2+, at the second site can activate the reaction with phosphorylase b, but the activity is inhibited by the binding of Mn2+ (10). Phosphorylase kinase acts on few other substrates, but it can phosphorylate a threonine residue in troponin I (16) and even inositol in phosphatidylinositol (17). We report in this communication that the binding of a second divalent cation in the recombinant catalytic subunit of phosphorylase kinase determines enzyme specificity. With Mg2+, seryl phosphorylation occurs, but with Mn2+ tyrosine phosphorylation is favored. Similar results are seen with the holoenzyme, Le. phosphorylase kinase has tyrosine kinase activity in the presence of Mn".
EXPERIMENTAL PROCEDURES '~a~eriu~s-HighIy purified rabbit skeletal muscle phosphorylase kinase was a kind gift from Dr. G. M. Carlson of the University of Tennessee. Recombinant truncated y subunit (1-300) of phosphorylase kinase is expressed, purified, and renatured as described by Huang et ai. (18). Phosphorylase b is prepared as described (19). Angiotensin I1 (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) is from Sigma. Phosphocellulose paper and ET-31 filter paper are from Whatman. ly-"P]ATP is the product of ICN. Other reagents used in experiments are reagent grade.
Protein Concentration Determination-The concentration of phosphorylase kinase and phosphorylase b is determined spectrophotometrically (20, 21). Angiotensin IT solution is prepared based on its dry weight. The concentration of truncated y subunit is determined by reaction with Bradford reagent {Bio-Rad).
Activity Assay-The seryl kinase activity of phosphorylase kinase is determined by measuring the incorporation of into phosphorylase b as described with some modification (18). The standards assay contained 50 mhi Tris, 50 m M PIPES, pH 8.2, 10 m M Mg", 10 mg/ml phosphorylase b, and 1 m M [y-"2PplATP. The final concentration of truncated y is adjusted to around 0.3 nM. The reaction is carried out at 30 "C and initiated by adding enzyme solution. After incubation a portion of reaction mixture is spotted on a square ET-31 filter paper, washed with trich~oroacetic acid, and analyzed by liquid scintillation counting (22). The tyrosine kinase activity is assayed using angiotensin I1 as a substrate. The reaction is performed in an assay mixture containing 10% glycerol, 50 m M Tris, 50 mbi PIPES, pH 7.9, 3 mkt MnC12, 2.5 m M angiotensin 11, and 1 m M [y-""PlATP. The final concentrations of the trun-P h o s p h o~~~a s e Kinase, the Dual Specificity Kinase cated y subunit and phosphorylase kinase were 10 and 100 pg/ml, respectively. The tyrosine kinase assay is allowed to proceed at 30 '"C for 30 min. After incubation, a portion of reaction mixture is spotted on the phosphocellulose paper and washed with cold 0.5% phosphoric acid. When using poly(Glu,Tyr) as substrate, the reaction mixture is handled as the serine kinase assay. Oxidation-The truncated y subunit is oxidized as essentially described by Landgraf et al. (23). To remove dithiothreitoi, the truncated y subunit is dialyzed against R buffer (10% glycerol, 50 m M Tris, 50 m M HEPES, 50 m M NaCl, pH 7.8) on Microdialyzer 500 (Bio-Rad) in the cold room for at least two buffer changes. The oxidation is carried out at room temperature in R buffer containing 0.1 mg/ml truncated y and an appropriate amount of Cu2+ (0.5-16 p~) for 20 min. After incubation, the oxidized truncated y subunits are subjected to seryl and tyrosyl kinases assay.
OZigo~ucleotide-direc~ed Site-specific ~Ut~genes~s-Mutagenesis is performed using an Amersham commercial kit as described previously (18). Mutation is identified by restriction enzyme analysis and verified hy sequencing.
Data Analysis-The kinetic data are analyzed with computer software Enzftter (Elsevier Science Publishers).

RESULTS AND DISCUSSION
Phosphory~ase kinase holoenzyme purified from rabbit skeletal muscle shows significant tyrosine kinase activity with angiotensin I1 in the presence of Mn2+ but not with Mg2+ (Fig. 1). Poly(Glu,~r) and Raytide, two other common tyrosine protein kinase substrates with a poly-negative charge, can be phosphorylated at a very low level. The tyrosine kinase activity of holoenzyme is affected little by 2 mM EGTA, a concentration that completely inhibits seryl phosphorylation (Fig. 11, suggesting that Ca2+ was not essential for tyrosyl phosphorylation.
Autophosphorylation on a and fl subunits of phosphorylase kinase is known to activate the enzyme C24-26). To determine whether preincubation of the enzyme with Ca2+, Mg2+, and ATP, a condition that causes autophosphorylation, can also activate its tyrosine kinase activity, the holoenzyme is first treated with the above reagents and then assayed for tyrosine kinase activity. Note that if the enzyme is not preincubated, there is a lag in the reaction (Fig. 2). After preincubation, the To investigate the tyrosine kinase activity of phosphorylase kinase more fully, a recombinant truncated form of catalytic subunit (1-300) of phosphorylase kinase was used. Any contamination of a bacterial tyrosine protein kinase in truncated y preparation is excluded by the fact that recombinant protein is renatured and purified to homogeneity as judged by SDS-polyacrylamide gel electrophoresis (18). The recombinant truncated y subunit also phosphorylates angiotensin 11. Mn2+ is required for the tyrosine kinase activity, although Go2+ can also stimulate a moderate (about 25% of tyrosine kinase activity with Mn2+) activity. In contrast, a little tyrosine kinase activity (1-5 8 ) can be seen by using Mg2+ alone. These results are consistent with the observation that most tyrosine kinases prefer Mn2+ more than Mg2+ for their activity (8). Poly(Glu,Tyr) and Raytide are poor substrates for truncated y. It is reasonable to suggest that both angiotensin I1 and phosphorylase can be recognized by a common set of determinants and hence a common active site because angiotensin I1 (Asp-Arg-Val-xr-Ile-His-Pro-Phe) shows some similarity in primary sequence to that of phosphorylase (Arg'o-Lys-Gln-Ile-Ser-Val-Arg-Gly-__

LeU'8).
It has been demonstrated that the serine/threonine kinase activity of the truncated y subunit can be inhibited by the oxidation of Cu2+ (27). The oxidation caused the formation of two pairs of disulfide bonds in or near the active site region and inactivated the conversion of phosphorylase b to a by interfering with the binding of substrates. If both serineithreonine and tyrosine kinase activities of phospho~lase kinase share the same active site in the y subunit, oxidation should interfere with the binding of both substrates. As shown in Fig. 3, both serineithreonine and tyrosine kinase activities of the truncated y subunit are inhibited identically with increasing Cu". The idea of two kinase activities sharing one active site is further supported by the result of site-directed mutagenesis at an invariant residue of the catalytic loop, Asp'5o corresponding to Asp166, the putative catalytic base, of CAMP-dependent protein kinase (28). A similar but uncharged residue, Asn, was used to replace Asp150 in y. The mutant is constructed, expressed, and purified to homogeneity. Both serinelthreonine and tyrosine kinase activities are abolished by this mutagenesis.
The influence of metal ions on the substrate specificity of the truncated y subunit of phosphorylase kinase is shown in Fig. 4.
Inhibition of serinekhreonine kinase activity and activation of tyrosine kinase activity of truncated y show parallel changes by increasing Mn2+ in the presence of a n equivalent amount of Mg2+ and ATP. The two activity curves intersect at a concentration of approximately 250 p~ Mn2+, which is also the ECEO for both curves, suggesting this effect is due to the binding of Mn2+ at a common site. The crystal structure of CAMP-dependent protein kinase (12) shows that the nucleotide binding site is located at the base of the small lobe, and the second metal ion and peptide substrate are mainly situated on the large lobe.  (4) suggested a flexible active site region to explain the dual specificity of some kinases. The two metal ions, Mn2+ and Mg", could stabilize different conformations because of differences in their chemical properties and ionic radii. These two metal ions interact differently in the nucleotide binding region of a mutant nitrogenase (33).
Kinetic results (Table I) show that in the presence of Mn2+ the K,,, for ATP (54 2 3 p~) is about the same as that measured in the presence of Mg2+ (79 p~) (18). The apparent K, for phosphorylase b (4.0 0.2 p~), however, is about 5-fold lower.
In the presence of Mg2+ the tyrosine kinase activity is almost non-detectable. The V, and K,,, are 0.47 2 0.02 nmoWmininmo1 and 4.4 2 0.2 mM, respectively, using angiotensin I1 as substrate in the presence of Mn2+. This result suggests that a conformational change at the active site region caused by the binding of Mn2+ may further expose the binding site to its substrate and gives more room for a phenolic group. It is known that the binding of a second metal ion can increase the affinity of enzyme for its substrate with the insulin receptor tyrosine kinase (34). A conformational change in y induced by Mn"+ may turn the alcoholic group of Serl* of phosphorylase b away from the putative catalytic base, Aspi50, and reduce the rate of phosphoryl transfer (apparent V, = 163 5 8 nmollmidnmol) ( Table I).
The tyrosine kinase activity of truncated y is similar to that of most tyrosine kinases. When compared with its serine kinase, the tyrosine kinase activity is about 9'350 of its serine kinase activity using MnATP as substrate ( Table I). This result is consistent with the general observation t,hat turnover rates of tyrosine protein kinases are usually several orders lower than that of serinehhreonine kinases.
So far, a t least 11 dual specificity kinases have been reported (4). Most of these enzymes, however, have been identified from autophosphorylation reactions targeting tyrosine and serine1 threonine residues or from reactivity with anti-phosphotyrosine antibodies after expression in bacteria. Some dual specificity kinases have been reported to phosphorylate exogenous substrates (5-7, 35, 36) and have a role in cell development.
The results presented herein show that phosphorylase kinase has dual specificity, but whether this new activity is physiologically significant remains to be determined.