N-myristylation of the catalytic subunit of cAMP-dependent protein kinase conveys structural stability

Coexpression of the yeast N-myristyltransferase with the murine catalytic subunit of cAMP-dependent protein kinase in prokaryotic cells results in the N-myristylation of the recombinant catalytic subunit. The acylated recombinant catalytic subunit was purified following in vitro holoenzyme formation with a mutant form of the regulatory subunit and compared to the non-myristylated recombinant enzyme and to the mammalian porcine enzyme. All three enzymes are very similar in terms of their kinetic properties and their capacity to reassociate in vitro with the regulatory subunit to form holoenzyme. In contrast, the myristylated recombinant catalytic subunit is significantly more stable to thermal denaturation than the non-myristylated enzyme. Its thermal stability is now comparable to the mammalian enzyme. All three catalytic subunits are significantly more stable to thermal denaturation when they are part of the holoenzyme complex. Each shows an increase in T1/2 of 10 degrees C. This study demonstrates that one function for the myristic acid at the NH2 terminus of the catalytic subunit is to provide structural stability.     

Ethanol causes translocation of cAMP-dependent protein kinase catalytic subunit to the nucleus

Short- and long-term ethanol exposures have been shown to alter cellular levels of cAMP, but little is known about the effects of ethanol on cAMP-dependent protein kinase (PKA). When cAMP levels increase, the catalytic subunit of PKA (C alpha) is released from the regulatory subunit, phosphorylates nearby proteins, and then translocates to the nucleus, where it regulates gene expression. Altered localization of C alpha would have profound effects on multiple cellular functions. Therefore, we investigated whether ethanol alters intracellular localization of C alpha. NG108-15 cells were incubated in the presence or absence of ethanol for as long as 48 h, and localization of PKA subunits was determined by immunocytochemistry. We found that ethanol exposure produced a significant translocation of C alpha from the Golgi area to the nucleus. C alpha remained in the nucleus as long as ethanol was present. There was no effect of ethanol on localization of the type I regulatory subunit of PKA. Ethanol also caused a 43% decrease in the amount of type I regulatory subunit but had no effect on the amount of C alpha as determined by Western blot. These data suggest that ethanol-induced translocation of C alpha to the nucleus may account, in part, for diverse changes in cellular function and gene expression produced by alcohol.     

The catalytic subunit of Dictyostelium cAMP-dependent protein kinase -- role of the N-terminal domain and of the C-terminal residues in catalytic activity and stability

The C subunit of Dictyostelium cAMP-dependent protein kinase (PKA) is unusually large (73 kDa) due to the presence of 330 amino acids N-terminal to the conserved catalytic core. The sequence following the core, including a C-terminal -Phe-Xaa-Xaa-Phe-COOH motif, is highly conserved. We have characterized the catalytic activity and stability of C subunits mutated in sequences outside the catalytic core and we have analyzed their ability to interact with the R subunit and with the heat-stable protein-kinase inhibitor PKI. Mutants carrying deletions in the N-terminal domain displayed little difference in their kinetic properties and retained their capacity to be inhibited by R subunit and by PKI. In contrast, the mutation of one or both of the phenylalanine residues in the C-terminal motif resulted in a decrease of catalytic activity and stability of the proteins. Inhibition by the R subunit or by PKI were however unaffected. Sequence-comparison analysis of other protein kinases revealed that a -Phe-Xaa-Xaa-Phe- motif is present in many Ser/Thr protein kinases, although its location at the very end of the polypeptide is a particular feature of the PKA family. We propose that the presence of this motif may serve to identify isoforms of protein kinases.     

A binary complex of the catalytic subunit of cAMP-dependent protein kinase and adenosine further defines conformational flexibility

BACKGROUND: cAMP-dependent protein kinase (cAPK), a ubiquitous protein in eukaryotic cells, is one of the simplest members of the protein kinase family. It was the first protein kinase to be crystallized and continues to serve as a biochemical and structural prototype for this family of enzymes. To further understand the conformational changes that occur in different liganded and unliganded states of cAPK, the catalytic subunit of cAPK was crystallized in the absence of peptide inhibitor. RESULTS: The crystal structure of the catalytic subunit of mouse recombinant cAPK (rC) complexed with adenosine was solved at 2.6 A resolution and refined to a crystallographic R factor of 21.9% with good stereochemical parameters. This is the first structure of the rC subunit that lacks a bound inhibitor or substrate peptide. The structure was solved by molecular replacement and comprises two lobes (large and small) which contain a number of conserved loops. CONCLUSIONS: The binary complex of rC and adenosine adopts an 'intermediate' conformation relative to the previously described 'closed' and 'open' conformations of other rC complexes. Based on a comparison of these structures, the induced fit that is necessary for catalysis and closing of the active-site cleft appears to be confined to the small lobe, as in the absence of the peptide the conformation of the large lobe, including the peptide-docking surface, does not change. Three specific components contribute to the closing of the cleft: rotation of the small lobe; movement of the C-terminal tail; and closing of the so-called glycine-rich loop. There is no induced fit in the large lobe to accommodate the peptide and the closing of the cleft. A portion of the C-terminal tail, residues 315-334, serves as a gate for the entry or exit of the nucleotide into the hydrophobic active-site cleft.     

Mutations in the catalytic subunit of the cAMP-dependent protein kinase interfere with holoenzyme formation without disrupting inhibition by protein kinase inhibitor

Three amino acids were identified in the catalytic (C) subunit of the cyclic AMP-dependent protein kinase that are involved in interaction with regulatory (R) subunit, but not with the specific protein kinase inhibitor, PKI. In a functional assay for gene induction, a C expression vector with serine or arginine substituted for Leu-198 and the double mutant C, His-87-->Gln/Trp-196-->Arg (Orellana, S. A., and McKnight, G. S. (1992) Proc. Natl. Acad. Sci, U.S.A. 89, 4726-4730), retained activity in the presence of an excess of RI or RII. In contrast, cotransfection of a full-length PKI expression vector completely inhibited the activity of both mutant and wild type C subunits. These data suggest that although the substrate/pseudosubstrate sites of R and PKI interact with C at the catalytic site, there is an additional domain on the C subunit that is involved in holoenzyme formation with R subunit and is distinct from sites specifying high affinity PKI binding.     

Crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with MgATP and peptide inhibitor

The structure of a ternary complex of the catalytic subunit of cAMP-dependent protein kinase, MgATP, and a 20-residue inhibitor peptide was determined at a resolution of 2.7 A using the difference Fourier technique starting from the model of the binary complex (Knighton et al., 1991a). The model of the ternary complex was refined using both X-PLOR and TNT to an R factor of 0.212 and 0.224, respectively. The orientation of the nucleotide and the interactions of MgATP with numerous conserved residues at the active site of the enzyme are clearly defined. The unique protein kinase nucleotide binding site consists of a five-stranded antiparallel beta-sheet with the base buried in a hydrophobic site along beta-strands 1 and 2 and fixed by hydrogen bonds to the N6 amino and N7 nitrogens. The small lobe secures the nucleotide via a glycine-rich loop and by ion pairing with Lys72 and Glu91. While the small lobe fixes the nontransferable alpha- and beta-phosphates in this inhibitor complex, the gamma-phosphate is secured by two Mg2+ ions and interacts both directly and indirectly with several residues in the large lobe--Asp184, Asn171, Lys168. Asp166 is positioned to serve as a catalytic base. The structure is correlated with previous chemical evidence, and the features that distinguish this nucleotide binding motif from other nucleotide binding proteins are delineated.     

Glutamic acid 203 of the cAMP-dependent protein kinase catalytic subunit participates in the inhibition by two isoforms of the protein kinase inhibitor

Although the protein kinase inhibitors (PKIs) are known to be potent and specific inhibitors of the catalytic (C) subunit of cAMP-dependent protein kinase, little is known about their physiological roles. Glutamate 203 of the C alpha isoform (C alpha E203) has been implicated in the binding of the arginine 15 residue of the skeletal isoform of PKI (PKI alpha R15) (Knighton, D. R., Zheng, J., Ten Eyck, L. F., Xuong, N., Taylor, S.S., and Sowadski, J. M. (1991) Science 253, 414-420). To investigate the role of C alpha E203 in the binding of PKI and in vivo C-PKI interactions, in vitro mutagenesis was used to change the C alpha E203 codon of the murine C alpha cDNA to alanine and glutamine codons. Initially, the C alpha E203 mutant proteins were expressed and purified from Escherichia coli. C alpha E203 is not essential for catalysis as all of the C subunit mutants were enzymatically active. The mutation of Glu203 did increase the apparent Km for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) severalfold but did not affect the apparent Km for ATP. The Vmax(app) was not affected by the mutation of C alpha E203. The mutation of C alpha E203 compromised the ability of PKI alpha (5-24), PKI alpha, and PKI beta to inhibit phosphotransferase activity. PKI alpha was altered using in vitro mutagenesis to probe the role of Arg15 in interacting with C alpha E203. The PKI alpha R15A mutant was reduced in its inhibition of C alpha. Preliminary studies of the expression of these C alpha mutants in COS cells gave similar results. These results suggest that the C alpha E203 mutants may be useful in assessing the role of PKI in vivo.     

Cloning of the pka1 gene encoding the catalytic subunit of the cAMP-dependent protein kinase in Schizosaccharomyces pombe

We have isolated Schizosaccharomyces pombe genes that confer sterility to the fission yeast cell when expressed from a multicopy plasmid. One of these genes strongly hybridized to a probe carrying the open reading frame of Saccharomyces cerevisiae TPK1, which encodes a catalytic subunit of the cAMP-dependent protein kinase (protein kinase A). This S. pombe gene, named pka1, has a coding potential of 512 amino acids, and the deduced gene product is 60% identical with the S. cerevisiae Tpk1 protein in the C-terminal 320 amino acids. Disruption of pka1 slows cell growth but is not lethal. The resultant cells, however, are highly derepressed for sexual development, readily undergoing conjugation and sporulation in the absence of nitrogen starvation. They are, thus, phenotypically indistinguishable from the adenylyl cyclase-defective (cyr1-) cells previously characterized, except that the pka1- spores are retarded in germination, whereas the cyr1- spores are not. Disruption of pka1 is epistatic to a defect in cgs1, which encodes the regulatory subunit of protein kinase A. These results strongly suggest that the product of pka1 is a catalytic subunit of protein kinase A and, furthermore, that S. pombe has only one gene encoding it. This situation contrasts with the case of S. cerevisiae, in which three genes encode the catalytic subunits.     

Backbone flexibility of five sites on the catalytic subunit of cAMP-dependent protein kinase in the open and closed conformations

To develop an alternative approach to measure peptidyl backbone flexibility and to expand our understanding of the segmental flexibility of cAMP-dependent protein kinase (cAPK), the effect of protein kinase inhibitor peptide, PKIalpha(5-24), and MgATP on the mobility of fluorescein selectively conjugated to five sites on the catalytic subunit of cAPK was examined. Specifically, five full-length, single-site catalytic subunit mutants (K16C, K81C, I244C, C199A, and N326C) were prepared, and fluorescein maleimide was selectively attached to the side chains of each substituted cysteine or, in the case of the C199A mutant, to the unprotected native C343. The time-resolved anisotropy decay profiles of the five fluorescein maleimide-conjugated mutants were well fit to a biexponential equation. The fast rotational correlation times of the fluorescein conjugates ranged between 1.9 and 2.8 ns and were inversely correlated (r = -0.87) to the averaged crystallographic main-chain atom B factors around each site of conjugation. The slow correlation times ranged between 25 and 28 ns and were about the same magnitude as the value of 21 ns estimated from the Stokes-Einstein equation. The presence of MgATP and PKIalpha(5-24), which induces the closed conformation of cAPK, was associated with a reduction of the fast rotational correlation time of the K81C conjugate, indicating that the peptidyl backbone around K81 is measurably less flexible when the C subunit is in the closed compared with the open conformation. The results suggest (i) that time-resolved fluorescence anisotropy can assess the nanosecond flexibility of short segments of the peptidyl backbone around each site of labeling and (ii) that the substrate/pseudosubstrate binding differentially affects the backbone flexibility of cAPK.     

Pathways for degradation of the catalytic subunit of cAMP-dependent protein kinase differ in wild-type and kinase-negative S49 mouse lymphoma cells

The catalytic subunit of cAMP-dependent protein kinase radiolabeled with [35S]methionine in wild-type S49 mouse lymphoma cells was degraded with half-lives of approximately 9.2 h in unstimulated cells and approximately 4.5 h in cells stimulated with a membrane-permeable cAMP analog. Turnover in kinase-negative mutant cells was about three times faster than in stimulated wild-type cells and appeared to involve a unique 47-kDa intermediate. Levels of catalytic subunit protein revealed by Western immunoblotting were consistent with the measured differences in turnover, but whereas the protein was mostly soluble in wild-type cell extracts, it was almost entirely insoluble in the mutant cell extracts. A substantial fraction of the catalytic subunit labeled in a 5-min pulse was soluble in kinase-negative cell extracts, but most of this material was rendered insoluble by incubating the cells for an additional 30 min before extraction. Degradation of the catalytic subunit in kinase-negative, but not in wild-type, cells was inhibited strongly by two specific peptide aldehyde inhibitors of the proteasomal chymotrypsin-like activity. An inhibitor of the proteasomal protease that prefers branched-chain amino acids had less of an effect on catalytic subunit degradation in the mutant cells.     

Expression of the catalytic subunit of cAMP-dependent protein kinase in Escherichia coli: multiple isozymes reflect different phosphorylation states

The catalytic subunit of mouse cAMP-dependent protein kinase expressed in Escherichia coli was separated into three distinct species using Mono-S ion exchange chromatography. These isoenzymes corresponded to three isoelectric variants with pIs of 6.4 (30%), 7.2 (60%) and 8.2 (10%). The Stokes' radius of each form was 27.7, 27.1 and 26.3 A respectively. Using electrospray mass spectroscopy the differences between the isozymes were shown to be due to phosphorylation, with each form differing by 80 mass units corresponding to a single phosphate. The fully phosphorylated recombinant enzyme contained four phosphates while the dominant isozyme contained only three. Since the enzyme is not phosphorylated when active site mutations are introduced into the C-subunit, these phosphates are incorporated in an autocatalytic mechanism and are not due to E. coli protein kinases. When the recombinant enzyme was compared with the mammalian porcine heart enzyme significant differences in post-translational modifications were observed. The mammalian enzyme could also be separated into two isozymes. However, in contrast to the recombinant enzyme, the mammalian isozymes displayed an identical mass of 40 840. This correlated with two different post-translational modifications: two phosphates and an N-terminal myristyl moiety. The importance of post-translational modifications, and in particular the phosphorylation state, for the expression of eukaryotic proteins in E. coli is discussed.     

Dimerization/docking domain of the type Ialpha regulatory subunit of cAMP-dependent protein kinase. Requirements for dimerization and docking are distinct but overlapping

Based on increasing evidence that the type I R subunits as well as the type II R subunits localize to specific subcellular sites, we have carried out an extensive characterization of the stable dimerization domain at the N terminus of RIalpha. Deletion mutants as well as alanine scanning mutagenesis were used to delineate critical regions as well as particular amino acids that are required for homodimerization. A set of nested deletion mutants defined a minimum core required for dimerization. Two single site mutations on the C37H template, RIalpha(F47A) and RIalpha(F52A), were sufficient to abolish dimerization. In addition to serving as a dimerization motif, this domain also serves as a docking surface for binding to dual specificity anchoring proteins (D-AKAPs) (Huang, L. J., Durick, K., Weiner, J. A., Chun, J., and Taylor, S. S. (1997) J. Biol. Chem. 272, 8057-8064; Huang, L. J., Durick, K., Weiner, J. A., Chun, J., and Taylor, S. S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11184-11189). A similar strategy was used to map the sequence requirements for anchoring of RIalpha to D-AKAP1. Although dimerization appears to be essential for anchoring to D-AKAP1, anchoring can also be abolished by the following single site mutations: C37H, V20A, and I25A. These sites define "hot spots" for the anchoring surface since each of these dimeric proteins are deficient in binding to D-AKAP1. In contrast to earlier predictions, the alignment of the dimerization/docking domains of RIalpha and RII show striking similarities yet subtle differences not only in their secondary structure (Newlon, M. G., Roy, M., Hausken, Z. E., Scott, J. D., and Jennings. P. A. (1997) J. Biol. Chem. 272, 23637-23644) but also in the distribution of residues important for both docking and dimerization functions.     

Quaternary structures of a catalytic subunit-regulatory subunit dimeric complex and the holoenzyme of the cAMP-dependent protein kinase by neutron contrast variation

Chimeric molecules of the cAMP-dependent protein kinase (PKA) holoenzyme (R2C2) and of a Delta1-91RC dimer were reconstituted using deuterated regulatory (R) and protiated catalytic (C) subunits. Small angle scattering with contrast variation has revealed the shapes and dispositions of R and C in the reconstituted complexes, leading to low resolution models for both forms. The crystal structures of C and a truncation mutant of R fit well within the molecular boundaries of the RC dimer model. The area of interaction between R and C is small, seemingly poised for dissociation upon a conformational transition within R induced by cAMP binding. Within the RC dimer, C has a "closed" conformation similar to that seen for C with a bound pseudosubstrate peptide. The model for the PKA holoenzyme has an extended dumbbell shape. The interconnecting bar is formed from the dimerization domains of the R subunits, arranged in an antiparallel configuration, while each lobe contains the cAMP-binding domains of one R interacting with one C. Our studies suggest that the PKA structure may be flexible via a hinge movement of each dumbbell lobe with respect to the dimerization domain. Sequence comparisons suggest that this hinge might be a property of the RII PKA isoforms.     

A conserved deamidation site at Asn 2 in the catalytic subunit of mammalian cAMP-dependent protein kinase detected by capillary LC-MS and tandem mass spectrometry

The N-terminal sequence myr-Gly-Asn is conserved among the myristoylated cAPK (protein kinase A) catalytic subunit isozymes Calpha, Cbeta, and Cgamma. By capillary LC-MS and tandem MS, we show that, in approximately one third of the Calpha and Cbeta enzyme populations from cattle, pig, rabbit, and rat striated muscle, Asn 2 is deamidated to Asp 2. This deamidation accounts for the major isoelectric variants of the cAPK C-subunits formerly called CA and CB. Deamidation also includes characteristic isoaspartate isomeric peptides from Calpha and Cbeta. Asn 2 deamidation does not occur during C-subunit preparation and is absent in recombinant myristoylated Calpha (rCalpha) from Escherichia coli. Deamidation appears to be the exclusive pathway for introduction of an acidic residue adjacent to the myristoylated N-terminal glycine, verified by the myristoylation negative phenotype of an rCalpha(Asn 2 Asp) mutant. This is the first report thus far of a naturally occurring myr-Gly-Asp sequence. Asp 2 seems to be required for the well-characterized (auto)phosphorylation of the native enzyme at Ser 10. Our results suggest that the myristoylated N terminus of cAPK is a conserved site for deamidation in vivo. Comparable myr-Gly-Asn sequences are found in several signaling proteins. This may be especially significant in view of the recent knowledge that negative charges close to myristic acid in some proteins contribute to regulating their cellular localization.     

Isolation and study of the properties of the catalytic subunit of cAMP-dependent protein kinase of the small intestinal mucosa of the rabbit

cAMP-dependent and casein proteinkinase were found in cytosol of the rabbit small intestine mucosa. cAMP-dependent proteinkinase of cytosol is represented by two forms of types I and II. The activity of enzymes of types I and II constitutes 10 and 90%, respectively. Casein proteinkinase is represented by a single form. The catalytic subunit of cAMP-dependent proteinkinase of type II was isolated in a homogenous state. The catalytic subunit phosphorylates histones H1, H2a, H2b and protamine and to a far less degree histones H3, H4 and casein (H2b greater than H1 greater than H2a greater than protamine much greater than H3 greater than casein). The Km value for histone H1 is equal to 65 mkM, and that for Mg-ATP 12 mkM. Chloromethylpyrophosphonate and adenosine p-fluorosulfobenzoate were studied as affine modifiers of the active center of the catalytic subunit from the small intestine mucosa. It was shown that only adenosine p-fluorosulfonate is an irreversible inhibitor of the catalytic subunit.     

Mitosis-specific phosphorylation and subcellular redistribution of the RIIalpha regulatory subunit of cAMP-dependent protein kinase

Phosphorylation of the RII regulatory subunits of cyclic AMP-dependent protein kinases (PKAs) was examined during the HeLa cell cycle. Three RIIalpha isoforms of 51, 54, and 57 kDa were identified by RIIalpha immunodetection and labeling with 8-azido[32P]cAMP in different cell cycle phases. These isoforms were characterized as different phosphorylation states by the use of selective PKA and cyclin-directed kinase inhibitors. Whereas RIIalpha autophosphorylation by PKA caused RIIalpha to shift from 51 to 54 kDa, phosphorylation of RIIalpha by one other or a combination of several kinases activated during mitosis caused RIIalpha to shift from 51 to 57 kDa. In vivo incorporation of [32P]orthophosphate into mitotic cells and RIIalpha immunoprecipitation demonstrated that RIIalpha was hyperphosphorylated on a different site than the one phosphorylated by PKA. Deletion and mutation analysis demonstrated that the cyclin B-p34(cdc2) kinase (CDK1) phosphorylated human recombinant RIIalpha in vitro on Thr54. Whereas RIIalpha was associated with the Golgi-centrosomal region during interphase, it was dissociated from its centrosomal localization at metaphase-anaphase transition. Furthermore, particulate RIIalpha from HeLa cell extracts was solubilized following incubation with CDK1 in vitro. Our results suggest that at the onset of mitosis, CDK1 phosphorylates RIIalpha, and this may alter its subcellular localization.     

Cryo-EM imaging of the catalytic subunit of the DNA-dependent protein kinase

The DNA-dependent protein kinase (DNA-PK) plays an important role in mammalian DNA double-strand break repair and immunoglobulin gene rearrangement. The DNA-PK holoenzyme is activated by assembly at DNA ends and is comprised of DNA-PKcs, a 460 kDa protein kinase catalytic subunit, and Ku, a 70 kDa/80 kDa heterodimeric DNA-targeting component. We have solved the three-dimensional structure of DNA-PKcs to approximately 21 A resolution by analytically combining images of nearly 9500 individual particles extracted from cryo-electron micrographs. The DNA-PKcs protein has an open, pseudo 2-fold symmetric structure with a gap separating a crown-shaped top from a rounded base. Columns of density are observed to protrude into the gap from both the crown and the base. Measurements of the enclosed volume indicate that the interior of the protein is largely hollow. The structure of DNA-PKcs suggests that its association with DNA may involve the internalization of double-stranded ends.     

Properties of the regulatory subunit of cAMP-dependent protein kinase type II from human brain

The regulatory subunit type II (RII) of cAMP-dependent protein kinase purified from human brain was represented by two proteins with apparent molecular masses of 51-52 kD and 54 kD. Dephosphorylation of human RII containing 3 mol phosphate/mol protein did not change the electrophoretic pattern. One-dimensional peptide mapping of 51-52 kD and 54 kD proteins after digestion with St. aureus V8 protease evidenced to their being distinct proteins. The data obtained permit to assume that human RII of neural type is represented by two isoforms.