Catalytic Subunit Of Phosphorylase Kinase Research Articles

Genome-wide association study identifies markers associated with meat ultimate pH in Duroc pigs.

In this study we aimed to identify genomic regions associated with muscle pH, meat colour and water-holding capacity in a population of 280 Italian Duroc pigs genotyped by the Illumina PorcineSNP60 v2 Genotyping BeadChip. After quality control, the remaining 32597 SNPs and 278 subjects were used to perform a genome-wide association study with the genabel package, using a kinship matrix in a model with the effects of sex, age and slaughter day. Bonferroni correction was applied, and the significant markers and regions were then further investigated to identify the nearest genes and the linkage disequilibrium (LD) between markers. Four markers (ASGA0082344, ASGA0095635, DBWU0000985 and CASI0005117) were significantly associated with ultimate pH (pHu ); no significant association was detected for the other traits. The four significant variants, located from 16.841 to 17.643Mb on chromosome 3, were found within or close to the sequences of the sulfatase modifying factor 2 (SUMF2), lysine acetyltransferase 8 (KAT8), serine protease 8 (PRSS8) and phosphorylase kinase catalytic subunit gamma 2 (PHKG2) genes. The four associated markers lie in two LD blocks, suggesting that the observed effect is related to mutations located in two regions: the first one where SUMF2 is mapped and the second one where genes KAT8, PRSS8 and PHKG2 are located.

Association between the splice mutation g.8283C>A of the PHKG1 gene and meat quality traits in Large White pigs

Pork technological and sensory qualities are greatly affected by water-holding capacity (WHC), pH, color, tenderness, and chemical composition, and the identification of genetic markers affecting these traits is regarded as a primary goal in pig selection. Phosphorylase Kinase Catalytic Subunit Gamma 1 (PHKG1) gene has been reported in the literature to be a candidate gene for meat drip loss, ultimate pH, color and glycolytic potential. This study was mainly aimed at testing whether PHKG1 g.8283C>A SNP was associated with meat quality traits in heavy pigs. The associations between PHKG1 g.8283C>A SNP, WHC, meat color and pH measurements were evaluated in 250 Italian Large White pigs. The marker showed an additive effect on WHC (P = .008) and a dominant effect on a* color parameter (P = .004). Further studies are needed to elucidate the effects of this polymorphism on heavy pig meat quality traits before this mutation could be considered a marker of interest for heavy pig selection schemes.

Structure and Location of the Regulatory β Subunits in the (αβγδ)4 Phosphorylase Kinase Complex

Phosphorylase kinase (PhK) is a hexadecameric (αβγδ)(4) complex that regulates glycogenolysis in skeletal muscle. Activity of the catalytic γ subunit is regulated by allosteric activators targeting the regulatory α, β, and δ subunits. Three-dimensional EM reconstructions of PhK show it to be two large (αβγδ)(2) lobes joined with D(2) symmetry through interconnecting bridges. The subunit composition of these bridges was unknown, although indirect evidence suggested the β subunits may be involved in their formation. We have used biochemical, biophysical, and computational approaches to not only address the quaternary structure of the β subunits within the PhK complex, i.e. whether they compose the bridges, but also their secondary and tertiary structures. The secondary structure of β was determined to be predominantly helical by comparing the CD spectrum of an αγδ subcomplex with that of the native (αβγδ)(4) complex. An atomic model displaying tertiary structure for the entire β subunit was constructed using chemical cross-linking, MS, threading, and ab initio approaches. Nearly all this model is covered by two templates corresponding to glycosyl hydrolase 15 family members and the A subunit of protein phosphatase 2A. Regarding the quaternary structure of the β subunits, they were directly determined to compose the four interconnecting bridges in the (αβγδ)(4) kinase core, because a β(4) subcomplex was observed through both chemical cross-linking and top-down MS of PhK. The predicted model of the β subunit was docked within the bridges of a cryoelectron microscopic density envelope of PhK utilizing known surface features of the subunit.

Isolation, expression patterns and SNP frequencies of the porcine PHKG2 gene

Phosphorylase kinase, gamma 2 gene ( PHKG2) encodes the catalytic subunit of phosphorylase kinase which is a regulatory enzyme in the cascade activation of glycogenolysis. In order to further understand the functions of the porcine PHKG2 gene, we obtained the complete open reading frame sequences and the determination of its expression by real-time PCR. Our spatial expression analysis showed that porcine PHKG2 gene was highly expressed in spleen, uterus and ovary. In contrast, low levels of expression of this gene were evident in heart, liver, lung, kidney, stomach, small intestine or fat, and much lower levels in skeletal muscle. Three SNPs, 785G>A, 866G>A and 875G>A were identified, their genotypes and allelic frequencies were analyzed. The frequencies of the G allele were dominant in Western pig breeds and the frequencies of the A allele were dominant in Chinese indigenous breeds in all analyzed polymorphisms. Haplotypes harboring these three polymorphisms were examined in five breeds, a total of 8 haplotypes were found in those breeds, which was equal to the theoretically expected number (2 3). The date presented here provided information about the functions of porcine PHKG2 gene.

Glucose Regulates EF-2 Phosphorylation and Protein Translation by a Protein Phosphatase-2A-dependent Mechanism in INS-1-derived 832/13 Cells

The role of elongation factor (EF)-2 phosphorylation in the regulation of pancreatic beta-cell protein synthesis by glucose was investigated in the INS-1-derived cell line 832/13. Incubation of cells in media containing 1 mm glucose resulted in a progressive increase in EF-2 phosphorylation that was maximal by 1-2 h. Readdition of 10 mm glucose promoted a rapid dephosphorylation of EF-2 that was complete in 10 min and maintained over the ensuing 2 h. Similar results were obtained using primary rat islets or Min-6 insulinoma cells. The glucose effect in 832/13 cells was replicated by addition of pyruvate or alpha-ketocaproate, but not 2-deoxyglucose, suggesting that mitochondrial metabolism was required. Accordingly, glucose-mediated dephosphorylation of EF-2 was completely blocked by the mitochondrial respiratory antagonists antimycin A and oligomycin. The hyperglycemic effect was not mimicked by incubation of cells in 100 nm insulin, 30 mm potassium chloride, or 0.25 mm diazoxide, indicating that insulin secretion and/or depolarization of beta cells was not required. The locus of the high glucose effect appeared to be protein phosphatase-2A, the principal phosphatase acting on EF-2. Protein phosphatase-2A activity was stimulated by glucose addition to 832/13 cells, but neither protein phosphatase-1 nor calmodulin kinase III (EF-2 kinase) activity was affected under these conditions. The slower rephosphorylation of EF-2 during the transition from high to low glucose may involve effects on EF-2 kinase activity. Addition of 5-aminoimidazole-4-carboxamide 1-beta-d-ribofuranoside in high glucose led to a marked stimulation of EF-2 phosphorylation, consistent with the possibility that increased AMP kinase activity in low glucose stimulates EF-2 kinase. In parallel with the effects on EF-2 dephosphorylation, addition of high glucose to 832/13 cells markedly increased the incorporation of [(35)S]methionine into total protein. Taken together, these results suggest that modulation of extracellular glucose impacts protein translation rate in beta cells at least in part through regulation of the elongation step, via phosphorylation/dephosphorylation of EF-2.

Differentiation-dependent mechanisms of transcriptional regulation of the catalytic subunit of phosphorylase kinase

The amount of phosphorylase kinase in skeletal muscle is exquisitely sensitive to developmental signals such as differentiation and innervation, and is clearly regulated in such a manner so as to always maintain the γ catalytic subunit under the control of its regulatory α, β and γ subunits. To identify how the transcription of the γ subunit is regulated, we have analysed 3.8kb of the upstream regulatory region using a luciferase reporter system. A complex sequence of interdependent regulations is evident. The γ catalytic subunit gene contains two inhibitory controls with very dominant features. Also evident are an array of multiple positive regulatory elements, prominent amongst which are four E-boxes, of which two are downstream, one is upstream and one is in the middle of the CAAT-TATA core promoter. Differentiation-dependent positive regulation arises as a consequence of both E-box regulation and the activation of at least one other regulatory element. The primary mode of transcriptional regulation of the γ catalytic subunit gene appears to occur by the relief of regulation of an otherwise default inhibitory status. It is noteworthy that such a mode of regulation mirrors the regulation of the enzymic activity of many protein kinases, including phosphorylase kinase. With phosphorylase kinase, both its transcriptional regulation as well as the regulation of the protein itself, are primed to maintain the γ catalytic subunit either unexpressed or inactivate respectively, until a positive signal occurs to override an otherwise dominant default inhibitory condition.

Differentiation-dependent mechanisms of transcriptional regulation of the catalytic subunit of phosphorylase kinase

The amount of phosphorylase kinase in skeletal muscle is exquisitely sensitive to developmental signals such as differentiation and innervation, and is clearly regulated in such a manner so as to always maintain the gamma catalytic subunit under the control of its regulatory alpha, beta and gamma subunits. To identify how the transcription of the gamma subunit is regulated, we have analysed 3.8 kb of the upstream regulatory region using a luciferase reporter system. A complex sequence of interdependent regulations is evident. The gamma catalytic subunit gene contains two inhibitory controls with very dominant features. Also evident are an array of multiple positive regulatory elements, prominent amongst which are four E-boxes, of which two are downstream, one is upstream and one is in the middle of the CAAT-TATA core promoter. Differentiation-dependent positive regulation arises as a consequence of both E-box regulation and the activation of at least one other regulatory element. The primary mode of transcriptional regulation of the gamma catalytic subunit gene appears to occur by the relief of regulation of an otherwise default inhibitory status. It is noteworthy that such a mode of regulation mirrors the regulation of the enzymic activity of many protein kinases, including phosphorylase kinase. With phosphorylase kinase, both its transcriptional regulation as well as the regulation of the protein itself, are primed to maintain the gamma catalytic subunit either unexpressed or inactivate respectively, until a positive signal occurs to override an otherwise dominant default inhibitory condition.

The regulatory alpha subunit of phosphorylase kinase may directly participate in the binding of glycogen phosphorylase.

The yeast two-hybrid screen has been used to identify potential regions of interaction of the largest regulatory subunit, alpha, of phosphorylase kinase (PhK) with two fragments of its protein substrate, glycogen phosphorylase b (Phb). One fragment, corresponding to residues 17-484 (PhbN'), contained the regulatory domain of the protein, but in missing the first 16 residues was devoid of the sole phosphorylation site of Phb, Ser14; the second fragment corresponded to residues 485-843 (PhbC) and contained the catalytic domain of Phb. Truncation fragments of the alpha subunit were screened for interactions against these two substrate fragments. PhbC was not found to interact with any alpha constructs; however, PhbN' interacted with a region of alpha (residues 864-1014) that is near the phosphorylatable region of that subunit. PhbN' was also screened for interactions against a variety of fragments of the catalytic gamma subunit of PhK; however, no interactions were detected, even with full-length gamma. Our results support the idea that amino acid residues proximal to the convertible serine of Phb are important for its specific interaction with the catalytic subunit of PhK, but that regions distinct from the convertible serine residue of Phb and from the catalytic domain of PhK may also be involved in the interaction of these two proteins.

An Inhibitory Segment of the Catalytic Subunit of Phosphorylase Kinase Does Not Act as a Pseudosubstrate

The C terminus of the catalytic gamma subunit of phosphorylase kinase contains two autoinhibitory calmodulin binding domains designated PhK13 and PhK5. These peptides inhibit truncated gamma(1-300). Previous data show that PhK13 (residues 302-326) is a competitive inhibitor with respect to phosphorylase b, with a K(i) of 1.8 microm. This result suggests that PhK13 may bind to the active site of truncated gamma(1-300). Variants of PhK13 were prepared to localize the determinants for interaction with the catalytic fragment gamma(1-300). PhK13-1, containing residues 302-312, was found to be a competitive inhibitor with respect to phosphorylase b with a K(i) of 6.0 microm. PhK13 has been proposed to function as a pseudosubstrate inhibitor with Cys-308 occupying the site that normally accommodates the phosphorylatable serine in phosphorylase b. A PhK13-1 variant, C308S, was synthesized. Kinetic characterization of this peptide reveals that it does not serve as a substrate but is a competitive inhibitor. Additional variants were designed based on previous knowledge of phosphorylase kinase substrate determinants. Variants were analyzed as substrates and as inhibitors for truncated gamma(1-300). Although PhK13-1 does not appear to function as a pseudosubstrate, several specificity determinants employed in the recognition of phosphorylase b as substrate are utilized in the recognition of PhK13-1 as an inhibitor.

Site-directed mutants of glycogen phosphorylase are altered in their interaction with phosphorylase kinase.

Glycogen phosphorylase is found in resting muscle as phosphorylase b, which is inactive without AMP. Phosphorylation by phosphorylase kinase (PhK) produces phosphorylase a, which is active in the absence of AMP. PhK is the only kinase that can phosphorylate phosphorylase b, which in turn is the only physiological substrate for PhK. We have explored the reasons for this specificity and how these two enzymes recognize each other by studying site-directed mutants of glycogen phosphorylase. All mutants were assayed for changes in their interaction with a truncated form of the catalytic subunit of phosphorylase kinase, gamma(1-300). Five mutations (R69K, R69E, R43E, R43E/R69E, and E501A), made at sites that interact with the amino terminus in either phosphorylase b or a, showed little difference in phosphorylation by gamma(1-300) compared to wild-type phosphorylase b. Five mutations, made at three sites in the amino-terminal tail of phosphorylase (K11A, K11E, I13G, R16A, and R16E), however, produced decreases in catalytic efficiency for gamma(1-300), compared to that for phosphorylase b. R16E was the poorest substrate for gamma(1-300), giving a 47-fold decrease in catalytic efficiency. The amino terminus, and especially Arg 16, are very important factors for recognition of phosphorylase by gamma(1-300). A specific interaction between Lys 11 of phosphorylase and Glu 110 of gamma(1-300) was also confirmed. In addition, I13G and R16A were able to be phosphorylated by protein kinase A, which does not recognize native phosphorylase.

Kinetic characterization of the double mutant R148A/E182S of glycogen phosphorylase kinase catalytic subunit: the role of the activation loop.

Many protein kinases are activated by phosphorylation in a highly conserved region of their catalytic subunit, termed activation loop. Phosphorylase kinase is constitutively active without the requirement for phosphorylation of residues in the activation loop. The residue which plays an analogous role to the phosphorylatable residues in other protein kinases is Glu182, which makes contacts to a highly conserved Arg148. In turn, Arg148 adjacent to the catalytic Asp149, enabling information to be transmitted from the activation loop to the catalytic machinery. The double mutant R148A/E182S has been kinetically characterized. The mutation resulted in an approximate 16- to 22-fold decrease in the kcat/Km value of the enzyme. The kinetic data, discussed in the light of the structural data from previously determined complexes of the enzyme, lead to the suggestion that the activation loop has a major role in substrate binding but also in correct orientation of the groups participating in catalysis.

Arginine to citrulline replacement in substrates of phosphorylase kinase

Synthetic peptides based on residues 9 to 18 of glycogen phosphorylase were prepared containing citrulline at position 10 or 16, or at both positions 10 and 16. The peptides were compared as substrates for a recombinant, truncated form of the catalytic subunit of phosphorylase kinase (residues 1–300). The peptide having citrulline at position 10 was phosphorylated the same as the parent peptide. Both the peptides with a single citrulline at position 16 and with two citrullines were phosphorylated less effectively than the parent peptide; k cat/ K m values were approximately 20% the value with the parent peptide. Incorporation of the second citrulline had little change in the effectiveness of the peptide as a substrate although the kinetic parameters with the citrulline peptides did show differences. The change in peptide phosphorylation did not seem to result from a change in peptide structure. Two-dimensional nuclear magnetic resonance studies of di-citrulline peptide are reported and showed no change in the solution structure of the peptide compared to the parent peptide. Thus, the change in kinetic parameters with the modified peptides seemed an effect of arginine replacement and was likely a consequence of the loss of charge inasmuch as the size of arginine and citrulline are similar. Arginine-16 was concluded to be more important for phosphorylase kinase recognition than arginine-10. These findings were consistent with the earlier studies using alanine replacement of arginine in synthetic peptides as substrates for the holoenzyme form of phosphorylase kinase.

Catalytic mechanism of phosphorylase kinase probed by mutational studies.

The contributions to catalysis of the conserved catalytic aspartate (Asp149) in the phosphorylase kinase catalytic subunit (PhK; residues 1-298) have been studied by kinetic and crystallographic methods. Kinetic studies in solvents of different viscosity show that PhK, like cyclic AMP dependent protein kinase, exhibits a mechanism in which the chemical step of phosphoryl transfer is fast and the rate-limiting step is release of the products, ADP and phosphoprotein, and possibly viscosity-dependent conformational changes. Site-directed mutagenesis of Asp149 to Ala and Asn resulted in enzymes with a small increase in K(m) for glycogen phosphorylase b (GPb) and ATP substrates and dramatic decreases in k(cat) (1.3 x 10(4) for Asp149Ala and 4.7 x 10(3) for Asp149Asn mutants, respectively). Viscosometric kinetic measurements with the Asp149Asn mutant showed a reduction in the rate-limiting step for release of products by 4.5 x 10(3) and a significant decrease (possibly as great as 2.2 x 10(3)) in the rate constant characterizing the chemical step. The date combined with the crystallographic evidence for the ternary PhK-AMPPNP-peptide complex [Lowe et al. (1997) EMBO J. 6, 6646-6658] provide powerful support for the role of the carboxyl of Asp149 in binding and orientation of the substrate and in catalysis of phosphoryl transfer. The constitutively active subunit PhK has a glutamate (Glu182) residue in the activation segment, in place of a phosphorylatable serine, threonine, or tyrosine residue in other protein kinases that are activated by phosphorylation. Site-directed mutagenesis of Glu182 and other residues involved in a hydrogen bond network resulted in mutant proteins (Glu182Ser, Arg148Ala, and Tyr206Phe) with decreased catalytic efficiency (approximate average decrease in k(cat)/K(m) by 20-fold). The crystal structure of the mutant Glu182Ser at 2.6 A resolution showed a phosphate dianion about 2.6 A from the position previously occupied by the carboxylate of Glu182. There was no change in tertiary structure from the native protein, but the activation segment in the region C-terminal to residue 182 showed increased disorder, indicating that correct localization of the activation segment is necessary in order to recognize and present the protein substrate for catalysis.

A Recombinant Form of the Catalytic Subunit of Phosphorylase Kinase That Is Soluble, Monomeric, and Includes Key C-Terminal Residues

Residues 302–326 of the catalytic (γ) subunit of phosphorylase kinase (PhK) may comprise an autoinhibitory, pseudosubstrate domain that binds calmodulin. To study this, the cDNA corresponding to rabbit muscle PhKγ was expressed using Escherichia coli. This yielded two stable, high-activity PhKγ forms (35 and 42 kDa by SDS–PAGE) that were smaller than an authentic sample of rabbit muscle PhKγ (45 kDa by SDS–PAGE). Each recombinant form was purified to homogeneity. The N-terminal sequence of the larger, 42-kDa form (pk42) matched that of the rabbit muscle enzyme. This suggested that pk42 consisted of PhKγ residues 1–362, including the putative calmodulin-binding, autoinhibitory domain. Kinetic parameters obtained for pk42 were like those previously reported for the intact γ subunit. This implied that the lack of 25 PhKγ C-terminal residues did not affect phosphorylase kinase activity, but greatly improved enzyme stability. An additional 60 residues were removed from the C-terminus of pk42 using the protease m-calpain. This increased the kinase activity 1.5-fold. Consistent with this, the activity of a mutant PhKγ that consisted of residues 1–300, denoted γ1–300, was like that of the m-calpain-treated enzyme. Therefore, although the effect was small, some influence by the C-terminus of pk42 was noted. Moreover, when pk42 was incubated with ATP alone, a C-terminal threonine residue became phosphorylated. Although the influence of this autophosphorylation cannot be inferred from this data, it was evidence that the C-terminus accessed the enzyme's active site. Taken together, these data imply that pk42 will be useful to study phosphorylase kinase structure/activity relationships.

Structural features contributing to complex formation between glycogen phosphorylase and phosphorylase kinase.

A polyclonal antibody was generated against a peptide corresponding to a region opposite the regulatory face of glycogen phosphorylase b (P-b), providing a probe for detecting and quantifying P-b when it is bound to its activating kinase, phosphorylase kinase (PhK). Using both direct and competition enzyme-linked immunosorbent assays (ELISAs), we have measured the extent of direct binding to PhK of various forms of phosphorylase, including different conformers induced by allosteric effectors as well as forms differing at the N-terminal site phosphorylated by PhK. Strong interactions with PhK were observed for both P-b', a truncated form lacking the site for phosphorylation, and P-a, the phosphorylated form of P-b. Further, the binding of P-b, P-b', and P-a was stimulated a similar amount by Mg(2+), or by Ca(2+) (both being activators of PhK). Our results suggest that the presence and conformation of P-b's N-terminal phosphorylation site do not fully account for the protein's affinity for PhK and that regions distinct from that site may also interact with PhK. Direct ELISAs detected the binding of P-b by a truncated form of the catalytic gamma subunit of PhK, consistent with the necessary interaction of PhK's catalytic subunit with its substrate P-b. In contrast, P-b' bound very poorly to the truncated gamma subunit, suggesting that the N-terminal phosphorylatable region of P-b may be critical in directing P-b to PhK's catalytic subunit and that the binding of P-b' by the PhK holoenzyme may involve more than just its catalytic core. The sum of our results suggests that structural features outside the catalytic domain of PhK and outside the phosphorylatable region of P-b may both be necessary for the maximal interaction of these two proteins.

Mg2+ induces conformational changes in the catalytic subunit of phosphorylase kinase, whether by itself or as part of the holoenzyme complex.

Phosphorylase kinase (PhK) from skeletal muscle is a structurally complex, highly regulated, hexadecameric enzyme of subunit composition (alpha beta gamma delta)4. Previous studies have revealed that the activity of its catalytic gamma subunit is controlled by alterations in quaternary structure initiated at allosteric and covalent modification sites on PhK's three regulatory subunits; however, changes in the conformation of the holoenzyme initiated by the catalytic subunit have been more difficult to document. In this study a monoclonal antibody (mAb gamma79) has been generated against isolated gamma subunit and used as a conformational probe of that subunit. The epitope recognized by this antibody is within the catalytic core of the gamma subunit, between residues 100 and 240, and monovalent fragments of the antibody inhibit the catalytic activity of the holoenzyme, the gamma-calmodulin binary complex, and the free gamma subunit. Activation of PhK by a variety of mechanisms known or thought to act through its regulatory subunits (phosphorylation, ADP binding, or alkaline pH) increased the binding of the holoenzyme to immobilized mAb gamma79, indicating that activation by any of these distinct mechanisms involves repositioning of the portion of the catalytic domain of the gamma subunit containing the epitope for mAb gamma79. The activating ligand Mg2+ also stimulated the binding of the PhK holoenzyme to immobilized mAb gamma79, as well as the binding of mAb gamma79 to immobilized gamma subunit. Thus, Mg2+ increases the accessibility of the mAb gamma79 epitope in both the isolated gamma subunit and in the holoenzyme. Our results suggest that previously reported influences of Mg2+ on the quaternary structure of the PhK holoenzyme are directly mediated by the gamma subunit.

The Testis Isoform of the Phosphorylase Kinase Catalytic Subunit (PhK-γT) Plays a Critical Role in Regulation of Glycogen Mobilization in Developing Lung

In order to identify the form of phosphorylase kinase catalytic subunit expressed in developing lung, degenerate polymerase chain reaction primers were designed based on conserved domains of the two known catalytic subunits, expressed primarily in muscle and testis. Amplification of cDNA from day 19 fetal rat lung followed by cloning and sequence analyses indicated that only the testis isoform of phosphorylase kinase (PhK-gammaT) was detectable in fetal lung. In situ hybridization analyses indicated that expression of PhK-gammaT RNA in developing lung tissue was widespread and not restricted to Type II epithelial cells; PhK-gammaT protein expression was temporally and spatially correlated with expression of PhK-gammaT RNA. PhK-gammaT RNA and protein expression was also characterized in the PhK-deficient glycogen storage disease (gsd) rat. PhK-gammaT RNA levels were similar in Type II cells isolated from wild type and gsd/gsd fetuses; in contrast, PhK-gammaT protein was virtually undetectable in gsd/ gsd Type II cells and enzyme activity was very low. These results suggest that PhK-gammaT plays a critical role in mobilization of glycogen during fetal lung development and that failure to catabolize glycogen in the gsd/gsd rat is related to an untranslatable PhK-gammaT RNA or unstable protein.

Oxidation and site-directed mutagenesis of the sulfhydryl groups of a truncated gamma catalytic subunit of phosphorylase kinase. Functional and structural effects.

A truncated form of the gamma subunit of phosphorylase kinase is inactivated by Cu2+ with the formation of two intra-molecular disulfide bonds. The formation of a disulfide bond between Cys-36 and Cys-172 (semioxidized form) results in approximately 50% loss of specific activity because the Km for MgATP is about 10-fold higher. The second disulfide bond is between Cys-184 and Cys-197 and causes further loss of activity. Eight Cys mutants, i.e. C36S, C36A, C42S, C138S, C172S, C184S, C184A, and C197S, were expressed and purified. Kinetic studies suggest that Cys-36 is important for interaction at the nucleotide site because of its hydrophobicity. With Cys-184 mutants, C184S and C184A, tyrosyl phosphorylation of angiotensin II is affected much more than serine kinase activity. The loss of tyrosine kinase activity is related to a lowered activity with Mn2+. With Mn2+, angiotensin II is a competitive inhibitor with respect to seryl kinase activity of C184S. With Mg2+, however, angiotensin II is a noncompetitive inhibitor. We suggest that metal ions influence the conformation of truncated gamma and that the protein substrate binding region containing Cys-184 is important for the dual specificity of this kinase.

Two exons encode the calmodulin-binding domain in the mouse phosphorylase kinase catalytic subunit gene

The catalytic subunit, γ, of phosphorylase kinase contains two calmodulin-binding sequences that define a domain in γ that is homologous to the troponin-C-binding domain in troponin I. The homology is based on both sequence and functional similarities. To account for this homology, it has been proposed that the calmodulin-binding sequences in γ and the troponin-C-binding domain in troponin I have evolved from a common ancestor. We investigated this possibility by comparing the exon structure of the γ gene with that of the troponin-I gene over their homologous domains. In the quail troponin-I gene, it is known that the entire troponin-C-binding domain is encoded by a single exon. However, two exons are found to encode the calmodulin-binding domain in the γ gene from mouse. This result indicates that convergent evolution may be responsible for the sequence and functional similarities between the homologous domains in troponin I and γ.

Features of calmodulin that are important in the activation of the catalytic subunit of phosphorylase kinase.

Calmodulin (CaM) is an integral subunit, called delta, of the phosphorylase kinase hexadecamer, and the activity of the isolated catalytic gamma-subunit of the kinase is stimulated by CaM. We report here the first analysis of functionally important features of CaM for activation of the gamma-subunit. A set of genetically engineered CaMs, in which acidic residues in each of the four E-helices of the "EF-hands" were changed to basic lysine residues, was used to probe the relative importance of charge features in each domain of CaM. The maximal activation of the isolated gamma-subunit was diminished by all of the charge reversal mutations. The gamma-subunit was especially sensitive to reversals in the second and third E-helix of CaM (residues 45-47 and 82-84), the latter being present in the central helix. The results suggest the functional importance of electrostatics in the interactions between the delta-subunit (CaM) and the catalytic gamma-subunit of phosphorylase kinase, which is similar to results obtained with CaM-dependent myosin light chain kinase (MLCK) from chicken gizzard and CaM-dependent protein kinase II (CaMPK-II). However, novel features of the interaction between CaM and the gamma-subunit of phosphorylase kinase are the significant contribution of electrostatics throughout the CaM molecule, including residues in both halves and on more than one face of CaM, and the lack of a major effect of the CaM mutations on substrate kinetic parameters, unlike the effects observed with MLCK and CaMPK-II. These results are consistent with a model in which the delta-subunit (CaM) of phosphorylase kinase interacts with an extended region or multiple regions of the gamma-subunit and suggest that the mechanism of CaM activation of the gamma-subunit may have features that are distinct from those of MLCK and CaMPK-II.