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J. Biol. Chem., Vol. 277, Issue 17, 14681-14687, April 26, 2002

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The Calmodulin-binding Domain of the Catalytic  Subunit of Phosphorylase Kinase Interacts with Its Inhibitory  Subunit

EVIDENCE FOR A Ca²⁺-SENSITIVE NETWORK OF QUATERNARY INTERACTIONS^*

Nancy A. Rice, Owen W. Nadeau, Qing Yang^§, and Gerald M. Carlson^¶

From the Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri, Kansas City, Missouri 64110-2499

Received for publication, February 6, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chemical cross-linking as a probe of conformation has consistently shown that activators, including Ca²⁺ ions, of the ()₄ phosphorylase kinase holoenzyme (PhK) alter the interactions between its regulatory  and catalytic  subunits. The  subunit is also known to interact with the  subunit, an endogenous molecule of calmodulin that mediates the activation of PhK by Ca²⁺ ions. In this study, we have used two-hybrid screening and chemical cross-linking to dissect the regulatory quaternary interactions involving these subunits. The yeast two-hybrid system indicated that regions near the C termini of the  (residues 343-386) and  (residues 1060-1237) subunits interact. The association of this region of  with  was corroborated by the isolation of a cross-linked fragment of  containing residues 1015-1237 from an dimer that had been formed within the PhK holoenzyme by formaldehyde, a nearly zero-length cross-linker. Because the region of  that we found to interact with  has previously been shown to contain a high affinity binding site for calmodulin (Dasgupta, M., Honeycutt, T., and Blumenthal, D. K. (1989) J. Biol. Chem. 264, 17156-17163), we tested the influence of Ca²⁺ on the conformation of the  subunit and found that the region of  that interacts with  was, in fact, perturbed by Ca²⁺. The results herein support the existence of a Ca²⁺-sensitive communication network among the , , and  subunits, with the regulatory domain of  being the primary mediator. The similarity of such a Ca²⁺-dependent network to the interactions among troponin C, troponin I, and actin is discussed in light of the known structural and functional similarities between troponin I and the  subunit of PhK.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Phosphorylase kinase (PhK)¹, a Ca²⁺-dependent enzyme involved in the regulation of glycogenolysis, is among the largest and most complex enzymes known. Structurally, PhK is composed of four copies each of four different subunits, ()₄ and has a mass of 1.3 × 10⁶ Da (for reviews see Refs. 1-3). Of the four subunits,  is catalytic, whereas the remaining three are regulatory:  and  exert quaternary constraint on the activity of , and  is an intrinsic molecule of calmodulin (CaM). To fully understand how PhK integrates diverse physiological signals to regulate glycogenolytic flux in skeletal muscle, it is first essential to understand how intrasubunit and intersubunit interactions within the hexadecameric holoenzyme change in response to effector ligands, and in so doing, control its catalytic activity. Despite the increased availability of structural information regarding PhK, interactions associated with activation and involving specific regions of individual subunits, in particular the  and  subunits, have largely remained uncharacterized. In this study, we have focused on delineating interacting regions between the large  and catalytic  subunits to advance our understanding of how structural perturbations correlate with activation of this complex holoenzyme.

By using chemical cross-linkers as structural probes, alterations in the interactions between the regulatory  and catalytic  subunits of PhK consistently emerge as a common structural marker of enzyme activation by multiple effectors, including Ca²⁺ ions (4-7). Although there have been many studies on the activation of PhK by Ca²⁺, it has not been clear how the binding of Ca²⁺ to the  subunit (CaM) relays structural information to the remainder of the holoenzyme, especially to the  subunit. It has been shown that Ca²⁺ increases the accessibility of specific regions of the  subunit (5), and the C-terminal region of the  subunit has been shown by a variety of experimental approaches to contain the regulatory domain that binds  (8-11), thus conferring Ca²⁺-sensitivity to the holoenzyme. In fact, truncation of  to eliminate this regulatory domain renders it constitutively active by itself and Ca²⁺/CaM-independent (9, 11). Our findings reported herein suggest that the flow of structural information from  to  in the holoenzyme is directly mediated by the C-terminal regulatory domain of . These are the first results to define a specific region of  that interacts with any region of either the  or  subunits of PhK. Furthermore, the finding that the region of - interaction includes a portion of the CaM binding domain of  provides a possible explanation for the previously observed changes in - interactions induced by Ca²⁺.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Yeast and Bacterial Strains-- Saccharomyces cerevisiae strain EGY48 (MAT, his3, trp1, ura3, LexAop-LEU2) (CLONTECH) was used for all two-hybrid analyses (12). All plasmid manipulations were performed according to standard protocol in the Escherichia coli strain DH5, unless otherwise specified (13, 14).

Proteins and Enzymes-- Nonactivated PhK was purified from fast-twitch skeletal muscle of New Zealand White rabbits (15), dialyzed against 50 mM Hepes (pH 6.8), 10% sucrose, and 0.2 mM EDTA and either used immediately or stored frozen at 80 °C. The full-length  subunit was purified from isolated holoenzyme by the method of Paudel and Carlson (16). Purified yeast and bovine brain CaM were generously provided by Drs. Trisha M. Davis (University of Washington) and Shengli Huang (University of Missouri Kansas City), respectively. Glycogen phosphorylase-b (P-b) was isolated from rabbit skeletal muscle as described (17), and residual AMP was removed with activated charcoal. The concentrations of the isolated  subunit and the two CaM isoforms were determined by the Bio-Rad protein assay with BSA as standard; P-b and PhK concentrations were determined spectrophotometrically by their respective absorbance indices (18, 19). Anti-PhK ,  and  subunit-specific mAbs were those previously described (20, 21). Anti-CaM was purchased from Signal Transduction Laboratories; all other detection conjugates were from Southern Biotechnology.

Two-hybrid Plasmid Construction-- All PhK  constructs were engineered as previously described (22). Rabbit skeletal muscle PhK  cDNA was kindly provided by Dr. Donald J. Graves (Iowa State University) and used as the template for the preparation of all  constructs. The cDNA fragments were ligated either to pLexA (CLONTECH), a 2-µ HIS3 plasmid, to generate a fusion protein consisting of the DNA BD (amino acids 1-202) of LexA fused to PhK  or to pB42AD (CLONTECH), a 2-µ TRP1 plasmid, to produce a B42 AD protein fused to PhK . Constructs 65C, 110C, and FL were directionally engineered into the EcoRI-BamHI restriction sites of pLexA by subcloning from previously prepared constructs in plasmid pGAD424. Subsequently, these three constructs were linearized from pLexA by digestion with EcoRI and XhoI restriction enzymes and ligated to pB42AD. Constructs 150C, 205C, 300C, and 342C were generated by PCR using primers to yield cDNAs flanked 5' and 3' with EcoRI and BamHI restriction sites, respectively (sense-strand, 5'-TGAATTCACCCGCGACGCGGCA-3'; antisense strand, (150) 5'-ATGGATCCTTACAGGTCCCGATGCAC-3', (205) 5'-ATGGATCCTTAGCCTGGGTGGTTGTC-3', (300) 5'-ATGGATCCTTAGGGGCTGAAGTGGC-3', (342) 5'-ATGGATCCTTACAGAGGTCGGAGGGC-3') and ligated to the EcoRI and BamHI sites of pLexA. To prepare the corresponding pB42AD  constructs, pLexA150C, pLexA205C, pLexA300C, and pLexA342C were digested with EcoRI and XhoI, and the corresponding linear  fragments were purified and subcloned into pB42AD. Fragments of  corresponding to its regulatory tail were generated by PCR using the following primers and ligated into both pLexA and pB42AD at EcoRI sites: sense strands, (301N) 5'-TGAATTCCGGGGGAAGTTCAAGGT-3' and (343N) 5'-TGAATTCCGCCGCCTCATCGACG-3'; antisense strand, 5'-TGAATTCTTAGTAGTCATCCTCAGCCAG-3'. The orientation and proper sequence of all LexA and B42  fusion proteins were verified by dideoxy sequencing and/or restriction mapping.

- Interactions by Two-hybrid Screening-- To screen for - interactions, two series of C-terminal deletion mutants of the  and  subunits were assayed for interactions as LexA and B42 fusion proteins in all possible binary combinations. Yeast strain EGY48, possessing the pSH18-34 lacZ reporter plasmid, was transformed by a modified lithium acetate procedure as previously described (23), and transformants were grown at 30 °C on synthetic medium lacking histidine, tryptophan, and uracil (SDHisTrpUra) for 3 days. Protein expression of all  and  constructs was verified by Western analysis, with minor modification, as previously described (24) using either a DNA BD cross-reactive LexA polyclonal Ab (kindly provided by Dr. Erica Golemis, Fox Chase Cancer Center) or an AD cross-reactive hemagglutinin mAb (Roche Molecular Biochemicals). Positive associations between  and  constructs were monitored by transcriptional activation of the LEU2 gene by growth on defined media lacking leucine (Leu) and of the lacZ reporter gene by liquid -galactosidase assays using o-nitrophenyl galactopyranoside as substrate (22, 25).

Two-hybrid Library Screening-- A rabbit skeletal muscle cDNA library (22) was used to screen for interactors of PhK by using 300C and FL constructs (described above) as bait. Yeast library transformants containing either of the DNA-BD bait plasmids and the AD cDNA library were generated as previously described (22). Transformation efficiencies of the library for both 300C and FL were 10⁴ colony-forming units/µg DNA with 9.5 × 10⁶ and 5.4 × 10⁶ total library clones eventually being amplified, respectively. All transformants were pooled and stored at 80 °C. Library screens were performed on SD/Gal/Raf (His, Trp, Ura, Leu) synthetic media in the presence of 80 µg/ml 5-bromo-4-chloro-3-indoyl--D-galactopyranoside in order to simultaneously screen for galactose-dependent LacZ expressing cDNA-encoded proteins that interact with either of the two bait proteins. After colonies were grown at 30 °C for 3-7 days, putative primary LacZ+/Leu+ colonies were restreaked onto SD His, Trp, Ura and subjected to secondary and tertiary analyses as previously described (22). Specific interactor cDNAs were ultimately identified by dideoxy sequencing.

Renaturation of the His-Trp Complex-- The isolated  subunit in 8 M urea, 0.1 M H₃PO₄, 0.1 mM EDTA, and 1 mM DTT was renatured as previously described (26, 27) in the presence of either yeast or bovine brain CaM. Each renaturation, carried out at 4 °C overnight, contained the following final concentrations: 0.125 µM isolated , 0.31-3.0 µM CaM, 1.66 mg/ml BSA, 0.8 M urea, 10 mM H₃PO₄, 0.1 mM EDTA, 0.5 mM CaCl₂, 0.3 mM DTT, and 100 mM Hepes (pH 8.0).

Enzymatic Assays of the /CaM Complex-- The activity of the renatured /CaM complex was determined by following the incorporation of ³²P into P-b at pH 7.0 using the filter paper assay of Reimann et al. (28). Prior to assaying, each renaturation sample was diluted 10-fold with buffer containing 100 mM Hepes (pH 7.0), 0.2 mM DTT, 0.5 mM CaCl₂, 0.1 mM EDTA, and 1 mg/ml BSA. To initiate the reaction, one volume of this diluted solution was added to three volumes of reaction mixture. Final concentrations in the standard assay were: 3.1 nM isolated , 0.67 mM CaCl₂, 10 mM Mg(CH₃CO₂)₂, 0.5 mM [-³²P]ATP, 100 mM Hepes (pH 7.0), 7.8-75 nM CaM, 0.3 mg/ml BSA, 2.8 mg/ml P-b, 20 mM urea, 25 µM EDTA, 0.75 µM DTT, and 0.25 mM H₃PO₄.

Cross-linking of PhK by Formaldehyde-- PhK was cross-linked by formaldehyde (5 mM), prepared by the hydrolysis of paraformaldehyde (29), either alone or in the presence of Ca²⁺. The final concentrations in the standard cross-linking reaction were: PhK (0.43 µM), Hepes (35 mM, pH 6.8), Ca²⁺ (1.25 mM), and EDTA (1 mM). Following cross-linking, reactions were quenched by an equal volume of SDS buffer (0.125 M Tris (pH 6.8), 20% glycerol, 5% -mercaptoethanol, 4% SDS). Cross-linked conjugates were resolved on SDS-PAGE gradient gels (4-20%) and characterized by their apparent mass and cross-reactivity against subunit-specific mAbs (20, 21).

Partial Proteolysis of Native, Ca²⁺-activated, and Cross-linked PhK-- PhK, either in the absence or presence of 1.25 mM Ca²⁺, was digested with chymotrypsin under conditions that promoted selective cleavage of the  subunit. The standard proteolysis reaction, containing 0.43 µM PhK, 0.12 µg/ml chymotrypsin, 39 mM Hepes (pH 6.8), and 1 mM EDTA, was carried out for 10 min at 30 °C and subsequently quenched by 2× SDS buffer with brief mixing and heating at 80 °C. Samples were resolved by SDS-PAGE on a 4-20% gradient gel, stained with Coomassie blue, and characterized by their apparent mass. The extent of  subunit digestion, which was linear over the time period used, and of the corresponding formation of proteolytic fragments were determined by optical integrated densitometry of the appropriate protein bands.

To localize the region of  cross-linked to  by formaldehyde, PhK was cross-linked as described above, but with the reaction quenched by addition of 1 M Tris (final concentration of 100 mM) and subsequently digested by chymotrypsin (0.5 µg/ml). The subunit composition of cross-linked species was determined by Western blotting and peptide sequencing. For sequencing, samples were electrophoretically transferred to polyvinylidene difluoride membranes in 10 mM 3-(cyclohexylamino)propanesulfonic acid (pH 11)/10% ethanol and stained with amido black for visualization. Bands of interest were excised and submitted for amino acid analysis and N-terminal sequencing to the Harvard Microchemistry Facility.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The C Terminus of PhK  Interacts with the Regulatory Domain of  in the Yeast Two-hybrid System-- Our laboratory has previously demonstrated the formation of - complexes through chemical cross-linking of the PhK holoenzyme (4-7); however, relatively long cross-linkers were used in those studies, and as a result, it was not unequivocally established that the  and  subunits within the hexadecameric holoenzyme actually interact, as opposed to simply being proximal. To determine whether the observed cross-linking of  with  results from the actual association of these two subunits, and if so, to define the regions involved in that interaction, we screened a series of C-terminal truncations of  and  (Fig. 1) against one another in the yeast two-hybrid system. To avoid potential disruption of secondary structural elements, the truncated mutants were designed based upon the known crystal structure of the catalytic domain of the  subunit (30) and the predicted secondary structure of the  subunit. When all binary combinations of the  and  constructs were assayed against each other, no interactions were observed for any C-terminal truncation of either  or ; a significant interaction did occur, however, when full-length constructs of both subunits were expressed (Table I). High levels of -galactosidase activity were induced by the interaction of FL and FL as either BD or AD fusions, but with the pair BD-FL/AD-FL demonstrating -galactosidase activity 3.4-fold greater than that observed with the reciprocal combination of constructs (133 versus 39.3 Miller units). Such domain effects are not uncommon in two-hybrid screens and have been observed with many proteins, including the transcriptional regulators Myc and Max (31).

View larger version (29K): [in this window] [in a new window]   Fig. 1.   PhK  and  subunit deletion mutants. A, PhK  deletion mutants. Constructs were engineered as either transcriptional DNA BD LexA or AD BD42 fusion proteins as described under "Experimental Procedures." Amino acid numbers are given above the first construct. Constructs are named as subunit followed by either the last C-terminal residue expressed beginning from residue 1 (e.g. 305C is a fusion protein of amino acids 1-305 of the  subunit) or by the first N-terminal residue expressed ending with the final amino acid of  (1237) (e.g. 1015N is a fusion protein of amino acids 1015-1237 of ). Functionally significant domains are represented by the various patterns as follows: gray, region missing in ' (50); hatched, region missing in  (51); and black and white, leucine zipper. B, PhK  deletion mutants. Constructs were engineered as described under "Experimental Procedures" and labeled as described in A. Sequence domains are represented by the various patterns as follows: checkered, unique N-terminal region; gray, hinge region; hatched, C-terminal regulatory domain; and black, CaM binding domains.

View this table: [in this window] [in a new window]   Table I Domain mapping of  and  interactions -Galactosidase activity from yeast lysates was determined using nitrophenyl galactopyranoside as substrate. Activity is expressed as Miller units according the following formula: A420 × 1000/(volume)(time)(A600). Data represent the mean ± S.E. of three assays performed in duplicate. A positive interaction is determined as being significantly greater than all control values. NT = not tested.

The fact that none of the C-terminal deletions, but only the full-length  and  constructs, interacted suggested that regions near the C termini of both subunits (residues 1060-1237 of  and 343-386 of ) are those that interact to form the - complex. To test this hypothesis, two constructs comprising either the entire C-terminal tail of , amino acids 301-386 (301N), or the shorter region therein implicated by deletion analysis to interact with , amino acids 343-386 (343N), were engineered as both DNA BD and AD fusion constructs and screened against all  subunit constructs (Fig. 1). Significant interactions were observed only between the 343N construct and the full-length  subunit, as either DNA BD or AD fusion proteins (14.0 and 23.6 Miller units, Table I) agreeing with the implied region of association between  and  from the truncation analyses. The reason why the longer  construct (301N) did not interact with any  construct is unclear. It should be noted, however, that in interpreting two-hybrid data, each BD and AD fusion protein is unique and must be evaluated as such; for instance, it is possible that the longer construct, but not the shorter, resulted in a chimeric protein structure in which the interaction of LexA with its targets was sterically blocked. Similar to the results obtained with 301N, when a construct expressing only the C terminus of  (residues 1015-1237 (1015N)) was screened as both a DNA BD and AD fusion protein against all of the  constructs shown in Fig. 1, no interactions were observed with any  mutant regardless of the transcriptional domain (data not shown). However, as is described below, a slightly shorter 1031N construct was found to interact with FL.

Additional evidence that the C-terminal CaM-binding regulatory domain of the  subunit interacts with the C-terminal region of the regulatory  subunit came from screening a rabbit skeletal muscle cDNA library (22) using FL versus 300C BD fusions as bait. Of ~6 × 10⁷ library transformants screened with FL, 165 primary LacZ+/Leu+ colonies were obtained. Of these initial positives, 63 were selected for further analysis, and 21 library cDNAs were ultimately sequenced. The sequencing results showed every single one of these 21 clones to be overlapping transcripts of the  subunit of PhK containing its entire C terminus, but varying in N-terminal start sites (Table II). Screening the very same cDNA library using 300C as bait resulted in 137 primary LacZ+/Leu+ library cDNAs, with 23 eventually being sequenced. All but two of these 23 were independent clones, but not a single one of them corresponded to the  subunit of PhK. Thus, elimination of the terminal 86 residues of  eliminated the  subunit as a target in the cDNA library, further demonstrating that in the two-hybrid system the C-terminal regulatory region of the  subunit of PhK interacts with the C-terminal region of its regulatory  subunit.

View this table: [in this window] [in a new window]   Table II Positive library  clones detected by FL-BD A rabbit skeletal muscle cDNA library was screened in the yeast two-hybrid system using full length  subunit of PhK as bait as described under "Experimental Procedures." Positive clones were sequenced and determined to be multiple overlapping C terminus transcripts of PhK .

Considering that a CaM-binding regulatory region of  (8) overlaps the region implicated above to bind  and that -- complexes can be isolated following partial dissociation of the rabbit muscle ()₄ holoenzyme (32) raised the question of whether, during our two-hybrid screening, endogenous yeast CaM, despite its structural and functional divergence from mammalian CaM (33, 34), may nevertheless interact with , giving rise to formation of ternary (--CaM), as opposed to binary (-), complexes. To evaluate the feasibility of this notion, we determined whether yeast CaM could bind and subsequently activate PhK . When the isolated  subunit of PhK was renatured in the presence of either purified yeast CaM or bovine brain CaM, the yeast CaM stimulated the P-b conversion activity of  to a similar extent as that observed with the bovine brain isoform. The concentrations for half-maximal activation by the yeast and bovine brain CaMs were very similar (K_a = 0.42 ± 0.18 µM, n = 5 versus K_a = 0.30 ± 0.10 µM, n = 3, respectively), the maximal activation by the yeast CaM was only 15% less than by the mammalian CaM (Fig. 2). These results indicate that in yeast, free CaM is capable of binding to  with high affinity; thus, it is possible that the observed - interactions identified in the two-hybrid system may actually involve yeast CaM also and correspond to interactions occurring within the -- trimer, assuming of course that  and CaM do not exclusively compete for binding to the C-terminal region of .

View larger version (9K): [in this window] [in a new window]   Fig. 2.   Stimulation of isolated  subunit catalytic activity by CaM. PhK  was renatured in the presence of either yeast () or bovine brain () CaM and assayed for enzymatic activity as described under "Experimental Procedures." Data points represent the average of triplicate assays with the bars indicating the S.D. Kinetic parameters given in the text were determined by a linear regression analysis of at least three to five separate experiments.

Formaldehyde Cross-links the C Terminus of the  Subunit to the  Subunit within the PhK Holoenzyme-- To further examine the contact regions between the  and  subunits, we sought a very short cross-linker that would enable us to substantiate the observed two-hybrid - interactions within the context of the ()₄ holoenzyme and, additionally, to correlate observed conformational changes between the two subunits with the activation of PhK. Formaldehyde was chosen as the cross-linking agent because its reaction with nucleophiles results in the insertion of but a single methylene bridge (~2.9 Å, Ref. 35) between reactive side chains. When PhK was cross-linked by formaldehyde, one major conjugate with an apparent mass of 178 kDa (based on sequences an - dimer has a theoretical mass of 183 kDa) was formed in large amounts (Fig. 3A). The cross-linking was determined to be intramolecular, i.e. within a PhK hexadecamer because the cross-linked protein coeluted with native, hexadecameric PhK on size exclusion high pressure liquid chromatography (data not shown), a method that we routinely use to determine the molecularity of cross-linking (36, 37). The formation of this 178-kDa conjugate, as followed by change in optical density, increased with time along with a corresponding loss in density of both the  and  subunits. Furthermore, the cross-linked species cross-reacted with only anti- and anti- subunit-specific mAbs by Western analysis; no cross-reactivity was observed with the anti- or anti-CaM () mAbs (Fig. 4B). Taken together, these data indicate the formation of an - dimer within the PhK holoenzyme by the very short cross-linker formaldehyde.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Ca2+-induced changes of the conformation of PhK. A, PhK (lane 1) was cross-linked by formaldehyde for 30 min in the absence (lane 2) or presence of 250 µM free Ca2+ (lane 3) and resolved by SDS-PAGE. B, optical density measurements of the Coomassie-stained - conjugates from lanes 2 and 3 of A were determined on a BioImage whole band analyzer. Bars represent the mean of three experiments ± S.E.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Partial proteolysis of native and cross-linked PhK by chymotrypsin. A, native or cross-linked PhK was partially digested with chymotrypsin, resolved by SDS-PAGE and stained for protein with Coomassie. Samples are as follows: 1, native PhK; 2, native PhK digested with chymotrypsin; 3, formaldehyde cross-linked PhK; 4, formaldehyde cross-linked PhK digested with chymotrypsin. The 58- and 24-kDa C-terminal fragments of  are indicated by arrows. B, parallel samples were transferred to nitrocellulose and probed with anti-, anti-, anti-, and anti-CaM mAbs as described under "Experimental Procedures." Sample order is identical to that of A. The two new bands corresponding to -1 (102 kDa) and -2 (68 kDa) formed by partial digestion of cross-linked PhK are labeled. A high molecular weight, anti- cross-reactive band is observed for native PhK digested by chymotrypsin and is most likely derived from an - dimer in which the  subunit is cross-linked to an N-terminal proteolytic fragment of  (36); a small amount of cross-linking of the holoenzyme by exposure to ultraviolet light, which occurs during typical handling and storage, has previously been observed (52).

To localize regions of the  subunit cross-linked to the  subunit, we relied on the highly selective proteolysis of the  subunit within the holoenzyme by chymotrypsin, which degrades that subunit essentially to completion without significant hydrolysis of the , , or  subunits (38). Partial digestion of nonactivated PhK generated major fragments of  having apparent masses of 78, 60, 58, 30, and 24 kDa; two fragments of  (58 and 24 kDa, Fig. 4A) have previously been shown to cross-react with an anti- mAb whose epitope is known to be near the C terminus of that subunit between residues 1132-1237 (7, 20). When cross-linked enzyme was digested with chymotrypsin, two new bands were observed, -₁ (102 kDa) and -₂ (68 kDa), which cross-reacted with both the anti- and anti- mAbs (Fig. 4B). Based upon mass, these new bands corresponded to the entire  subunit cross-linked to the 58- and 24-kDa C-terminal fragments of , respectively. Examination of the proteolytic digestion pattern of cross-linked PhK demonstrated corresponding losses in density of both the 58- and 24-kDa fragments of  in the anti- blot, as well as a significant loss in density of the  subunit in the anti- blot with respect to uncross-linked proteolysed holoenzyme.

Because -₂ was the better resolved of the two cross-linked - complexes and was present in greater quantities than -₁, it was used for subsequent sequence determination. N-terminal analysis of this conjugate resulted in two sequences being identified, TRDAALPG and RRLSISTE, which correspond exclusively to the N termini of  and the 24-kDa fragment of , respectively. The latter is known to result from cleavage at Phe-1014 and correspond to residues 1015-1237 of  (20), which make up the C-terminal one-sixth of the  subunit.

Effects of Ca²⁺ on the Conformational Stability of the  and  Subunits-- Ca²⁺ is the most fundamental activator of PhK, regulating its activity through the  subunit, an endogenous molecule of CaM. Because the C terminus of , known to bind to  (8-11), was identified in this study to also associate with , we investigated the effect of Ca²⁺ on - dimer formation by formaldehyde. The extent of - dimer produced by cross-linking the holoenzyme in the presence of Ca²⁺ increased by 2-fold over nonactivated PhK (Fig. 3). This enhancement of the formation of - by Ca²⁺ corroborates the observed two-hybrid interaction of  with the CaM-binding C terminus of  and is consistent with other cross-linking studies from our laboratory correlating Ca²⁺-mediated activation of the PhK holoenzyme with perturbations in the interaction between its  and  subunits (5-7).

Because the -mediated effects of Ca²⁺ resulting in the activation of  are concomitantly transmitted to the C-terminal region of , we further examined the Ca²⁺-induced perturbations in this region of  by its selective cleavage with chymotrypsin. When the PhK holoenzyme was partially digested in the presence of Ca²⁺, the pattern of cleavage of the  subunit did not change; however, there was a preferential 3-fold increase² in the rate of formation of the 24-kDa immunoreactive fragment of  previously shown to result from cleavage at residue 1014. These cross-linking and partial proteolysis results indicate that the binding of Ca²⁺ ions to the  subunit of PhK causes a distinct conformational change in the C-terminal region of its  subunit.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Whereas the catalytic  subunit of PhK is undoubtedly involved in complex interactions with all three of its regulatory subunits, no specific contact region between  and either of the large inhibitory  and  subunits has been determined previously. In this study, we have identified a region within the stretch of residues 343-386 at the C terminus of the  subunit that interacts with a region near the C terminus of the  subunit. This region of  is of particular importance because it contains one of the two distinct, noncontiguous CaM-binding domains present in the C-terminal regulatory region of the  subunit. In a thorough study utilizing a series of 18 overlapping 25-mer peptides corresponding to the C-terminal 110-residue sequence (amino acids 277-386) of the  subunit, Dasgupta et al. (8) identified two domains of  with nanomolar affinity for CaM: domain N (residues 287-331) and domain C (residues 332-371). Within domain C, the peptide with the highest affinity for CaM corresponded to residues 342-366, which but for one residue are contained entirely within the region of  shown herein to interact with .

The fact that the same region within the regulatory domain of the  subunit potentially interacts with both the  (CaM) and  subunits provides a plausible mechanism to explain how Ca²⁺ induces tertiary and quaternary structural changes that are associated with activation of the PhK holoenzyme. It has been previously suggested that activation of PhK by various effectors occurs via a hierarchy of tiered conformational changes, largely reflecting differing states of release of quaternary constraint imposed by the regulatory subunits upon the catalytic  subunit, with the most fundamental change being that induced by Ca²⁺ (discussed in detail in Ref. 5). By cross-linking analysis, it is known that structural perturbations involving the  and  subunits occur upon activation of PhK by the binding of Ca²⁺ ions to the  subunit (5-7). Therefore, it is reasonable to hypothesize that the binding of the C-terminal regulatory region of  to both  and  may be modulated in a Ca²⁺-dependent manner; that Ca²⁺ affects the conformation of the very region of  found to bind  (Fig. 4) supports this hypothesis. The evidence indicates that perturbation of the - interactions caused by the binding of Ca²⁺ to  occurs concomitant with perturbation of the - interactions, resulting in a -- communication network. Recently, three-dimensional structures of the holoenzyme obtained by image reconstruction of PhK particles observed by electron microscopy ± Ca²⁺ (39) revealed that Ca²⁺ does indeed induce distinct conformational changes over a region of PhK previously shown to be occupied by portions of its , , and  subunits (20, 21, 37).

Given that purified yeast CaM activates the isolated  subunit (Fig. 2), it is possible that endogenous CaM may be binding during our two-hybrid analyses to those  constructs that have a CaM-binding domain, and as a result, the positive interaction we observe between full-length  and  may actually represent a trimeric -- instead of a dimeric - complex. Typically, yeast CaM is a poor activator of mammalian target enzymes including protein kinases (34, 40), due to its significant divergence both structurally and functionally from mammalian CaMs; it shares only 60% identity in primary structure (33) and does not bind Ca²⁺ at site IV (41, 42). It is not surprising, however, that yeast CaM is capable of binding to and activating PhK  because the  subunit seems to be more sensitive to mutations in the second and third domains of CaM than in its 1st and fourth(43); moreover, the quite dissimilar TnC also activates , albeit to a lesser extent than CaM (44).

If our two-hybrid interactions do, in fact, involve not only  and , but also CaM, then additional issues arise concerning Ca²⁺ dependence and the interactions of the  subunit of PhK. Because the three stable species of PhK (the hexadecameric ()₄ holoenzyme, the -- trimer, and the - dimer) all differ in the extent to which their activity is dependent on Ca²⁺ ions, it has been suggested that increasing enzyme complexity, i.e. progressing from - to -- to holoenzyme, results in a progressive increase in the Ca²⁺-requirement for catalytic activation of the complex (32). Because the - complex readily dissociates in 8 M urea, whereas -- does not, it has been further suggested that extended interactions exist among these three subunits in which , besides binding , is stabilized from dissociation by the presence of  (45). The sum of these findings suggests that  may contribute, at least indirectly, to regulating the effects of Ca²⁺ on catalysis. Our current finding that the C terminus of , a regulatory region that undergoes structural perturbations in response to a variety of activating stimuli including Ca²⁺ (7), interacts with  within a region where it binds  supports the existence of an intricate, Ca²⁺-sensitive communication network among the , , and  subunits, an idea also strongly supported by the previously mentioned structures of PhK from electron microscopy (39). Because Ca²⁺-triggered communication can flow from the  to the  subunit, then it is reasonable to expect that some manifestation of the reverse process ought to be present, as well. This expectation may be born out by early reports that activation of the PhK holoenzyme by phosphorylation of its  and  subunits increases affinity of the  subunit for Ca²⁺ ions (46, 47).

Ca²⁺-dependent structural changes, similar to those described above for PhK, have been observed in the regulation of skeletal muscle troponin, specifically between the inhibitory region of TnI and its protein targets, actin and TnC (a CaM homologue). In reconstituted thin filaments, the inhibitory region of TnI, which shares remarkable sequence similarity with the C-terminal regulatory domain of the  subunit of PhK (8, 44), interacts preferentially with actin in the absence of Ca²⁺ but with TnC in the presence of Ca²⁺ (48). The possibility that the inhibitory domain of TnI and the homologous region within the  subunit of PhK share a similar mechanism in their interactions with their respective protein targets (TnC and actin versus the  and  subunits) is supported by the following striking structural and functional similarities. (a) The regions of greatest sequence identity between  and TnI (amino acids 301-325 and 103-115, respectively) contain in the case of TnI those very residues most critical for its specific interactions with actin and TnC (discussed in Ref. 44) and in the case of , one of its two adjacent CaM-binding domains (8). (b) TnC activates PhK  (44). (c) Actin inhibits -CaM and -TnC complexes (44). (d)  inhibits actomyosin ATPase (44). The  subunit of PhK is thought to have evolved from the fusion of a protein kinase protogene with a progenitor of exon VII of the TnI gene (44), which encodes its inhibitory domain. The probable evolutionary link between TnI and PhK , the homologies that exist between them, and our current findings suggest that in skeletal muscle the troponin complex and the PhK holoenzyme may share related Ca²⁺-dependent alterations in quaternary structure, all leading to the simultaneous stimulation by Ca²⁺ ions of contraction and energy production, respectively.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant DK32953 (to G. M. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Current address: Dept. of Molecular, Cellular, and Developmental Biology, University of Colorado, Campus Box 347, Boulder, CO 80309-0347.

^§ Current address: Dept. of Pediatrics, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160.

^¶ To whom correspondence should be addressed: Div. of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City, 503 Biological Sciences Bldg., 5100, Rockhill Rd., Kansas City, MO 64110-2499. Tel.: 816-235-2235; Fax: 816-235-5595; E-mail: carlsongm@umkc.edu.

Published, JBC Papers in Press, February 14, 2002, DOI 10.1074/jbc.M201229200

² Ca²⁺ causes a modest increase (~20%) in the activity of chymotrypsin (49). To control for this activation, the amount of the 24-kDa fragment generated from the  subunit was determined by densitometry and expressed as a percentage of the total  subunit consumed. During standard digestions, the percent of the 24-kDa fragment formed per total  consumed for Ca²⁺-activated PhK was 15.4% versus 4.9% for non-activated PhK.

	ABBREVIATIONS

The abbreviations used are: PhK, phosphorylase b kinase; CaM, calmodulin; P-b, glycogen phosphorylase-b; BSA, bovine serum albumin; mAb(s), monoclonal antibody(ies); BD, binding domain; AD, activation domain; DTT, dithiothreitol; TnI, troponin I; TnC, troponin C; FL, full-length; SD, synthetic-defined.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES