Biomedical subjects

S S Taylor

Publications and source records attributed to S S Taylor.

At least 55 records · Page 3Linked to original sources

PubMed1998

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.

Animals↗

PubMed1998

Phosphorylation and activation of cAMP-dependent protein kinase by phosphoinositide-dependent protein kinase.

Although phosphorylation of Thr-197 in the activation loop of the catalytic subunit of cAMP-dependent protein kinase (PKA) is an essential step for its proper biological function, the kinase responsible for this reaction in vivo has remained elusive. Using nonphosphorylated recombinant catalytic subunit as a substrate, we have shown that the phosphoinositide-dependent protein kinase, PDK1, expressed in 293 cells, phosphorylates and activates the catalytic subunit of PKA. The phosphorylation of PKA by PDK1 is rapid and is insensitive to PKI, the highly specific heat-stable protein kinase inhibitor. A mutant form of the catalytic subunit where Thr-197 was replaced with Asp was not a substrate for PDK1. In addition, phosphorylation of the catalytic subunit can be monitored immunochemically by using antibodies that recognize Thr-197 phosphorylated enzyme but not unphosphorylated enzyme or the Thr197Asp mutant. PDK1, or one of its homologs, is thus a likely candidate for the in vivo PKA kinase that phosphorylates Thr-197. This finding opens a new dimension in our thinking about this ubiquitous protein kinase and how it is regulated in the cell.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed1998

Endogenous protein kinase A inhibitor (PKIalpha) modulates synaptic activity.

Protein kinase A (PKA) has long been known to be involved in major regulatory mechanisms underlying synaptic plasticity and complex behaviors such as learning and memory. The endogenous PKA inhibitor, PKIalpha, has been extensively studied for its effects on PKA and PKA-mediated signal transduction. Clear functions for PKIalpha in vivo, however, remain to be established. Here we describe that several forms of synaptic stimulation in the rat hippocampus cause a dramatic decrease in the concentration of PKIalpha in dentate granule cells. Furthermore, chronic infusion of antisense oligonucleotides against PKIalpha into the rat brain results in a dramatic reduction of the excitability of these neurons and elimination of their ability to exhibit long-term potentiation (LTP) and long-term depression (LTD), suggesting a stimulus-dependent regulatory role for PKIalpha in PKA signal transduction.

Adaptor Proteins, Signal Transducing↗

PubMed1998

The human homologue of Bub3 is required for kinetochore localization of Bub1 and a Mad3/Bub1-related protein kinase.

A feedback control mechanism, or cell cycle checkpoint, delays the onset of anaphase until all the chromosomes are correctly aligned on the mitotic spindle. Previously, we showed that the murine homologue of Bub1 is not only required for checkpoint response to spindle damage, but also restrains progression through a normal mitosis (Taylor, S.S., and F. McKeon. 1997. Cell. 89:727-735). Here, we describe the identification of a human homologue of Bub3, a 37-kD protein with four WD repeats. Like Bub1, Bub3 localizes to kinetochores before chromosome alignment. In addition, Bub3 and Bub1 interact in mammalian cells. Deletion mapping was used to identify the domain of Bub1 required for binding Bub3. Significantly, this same domain is required for kinetochore localization of Bub1, suggesting that the role of Bub3 is to localize Bub1 to the kinetochore, thereby activating the checkpoint in response to unattached kinetochores. The identification of a human Mad3/Bub1-related protein kinase, hBubR1, which can also bind Bub3 in mammalian cells, is described. Ectopically expressed hBubR1 also localizes to kinetochores during prometaphase, but only when hBub3 is overexpressed. We discuss the implications of the common interaction between Bub1 and hBubR1 with hBub3 for checkpoint control.

Amino Acid Sequence↗

PubMed1998

Kinetic analyses of mutations in the glycine-rich loop of cAMP-dependent protein kinase.

The conserved glycines in the glycine-rich loop (Leu-Gly50-Thr-Gly52-Ser-Phe-Gly55-Arg-Val) of the catalytic (C) subunit of cAMP-dependent protein kinase were each mutated to Ser (G50S, G52S, and G55S). The effects of these mutations were assessed here using both steady-state and pre-steady-state kinetic methods. While G50S and G52S reduced the apparent affinity for ATP by approximately 10-fold, substitution at Gly55 had no effect on nucleotide binding. In contrast to ATP, only mutation at position 50 interfered with ADP binding. These three mutations lowered the rate of phosphoryl transfer by 7-300-fold. The combined data indicate that G50 and G52 are the most critical residues in the loop for catalysis, with replacement at position 52 being the most extreme owing to a larger decrease in the rate of phosphoryl transfer (29 vs 1.6 s-1 in contrast to 500 s-1 for wild-type C). Surprisingly, all three mutations lowered the affinity for Kemptide by approximately 10-fold, although none of the loop glycines makes direct contact with the substrate. The inability to correlate the rate constant for net product release with the dissociation constant for ADP implies that other steps may limit the decomposition of the ternary product complex. The observations that G52S (a) selectively affects ATP binding and (b) significantly lowers the rate of phosphoryl transfer without making direct contact with either the nucleotide or the peptide imply that this residue serves a structural role in the loop, most likely by positioning the backbone amide of Ser53 for contacting the gamma-phosphate of ATP. Energy-minimized models of the mutant proteins are consistent with the observed kinetic consequences of each mutation. The models predict that only mutation of Gly52 will interfere with the observed hydrogen bonding between the backbone and ATP.

Amino Acid Substitution↗

PubMed1998

Conserved water molecules contribute to the extensive network of interactions at the active site of protein kinase A.

Protein kinases constitute a large family of regulatory enzymes, each with a distinct specificity to restrict its action to its physiological target(s) only. The catalytic (C) subunit of protein kinase A, regarded as a structural prototype for this family, is composed of a conserved core flanked by two nonconserved segments at the amino and carboxyl termini. Here we summarize evidence to show that (i) the active site consists of an extended network of interactions that weave together both domains of the core as well as both segments that flank the core; (ii) the opening and closing of the active site cleft, including the dynamic and coordinated movement of the carboxyl terminal tail, contributes directly to substrate recognition and catalysis; and (iii) in addition to peptide and ATP, the active site contains six structured water molecules that constitute a conserved structural element of the active site. One of these active-site conserved water molecules is locked into place by its interactions with the nucleotide, the peptide substrate/inhibitor, the small and large domains of the conserved core, and Tyr-330 from the carboxyl-terminal "tail."

Animals↗

PubMed1998

Shc and Enigma are both required for mitogenic signaling by Ret/ptc2.

Ret/ptc2 is a constitutively active, oncogenic form of the c-Ret receptor tyrosine kinase. Like the other papillary thyroid carcinoma forms of Ret, Ret/ptc2 is activated through fusion of the Ret tyrosine kinase domain to the dimerization domain of another protein. Investigation of requirements for Ret/ptc2 mitogenic activity, using coexpression with dominant negative forms of Ras and Raf, indicated that these proteins are required for mitogenic signaling by Ret/ptc2. Because activation of Ras requires recruitment of Grb2 and SOS to the plasma membrane, the subcellular distribution of Ret/ptc2 was investigated, and it was found to localize to the cell periphery. This localization was mediated by association with Enigma via the Ret/ptc2 sequence containing tyrosine 586. Because Shc interacts with MEN2 forms of Ret, and because phosphorylation of Shc results in Grb2 recruitment and subsequent signaling through Ras and Raf, the potential interaction between Ret/ptc2 and Shc was investigated. The PTB domain of Shc also interacted with Ret/ptc2 at tyrosine 586, and this association resulted in tyrosine phosphorylation of Shc. Coexpression of chimeric proteins demonstrated that mitogenic signaling from Ret/ptc2 required both recruitment of Shc and subcellular localization by Enigma. Because Shc and Enigma interact with the same site on a Ret/ptc2 monomer, dimerization of Ret/ptc2 allows assembly of molecular complexes that are properly localized via Enigma and transmit mitogenic signals via Shc.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Dissecting the cooperative reassociation of the regulatory and catalytic subunits of cAMP-dependent protein kinase. Role of Trp-196 in the catalytic subunit.

The catalytic (C) subunit of cAMP-dependent protein kinase requires two distinct surfaces to form a stable complex with its physiological inhibitors, the regulatory (R) subunits and the heat-stable protein kinase inhibitors. In addition to a substrate-like segment that is common to both inhibitors, R requires a peripheral recognition site, PRS2. This surface is comprised of the essential phosphorylation site, Thr-197, His-87, Trp-196, and several surrounding basic residues. To probe the role of Trp-196 in the recognition of R, Trp-196 was replaced with Arg and Ala. Although both rC(W196A) and rC(W196R) were inhibited readily with cAMP-free R, they failed to form an inhibited holoenzyme complex with native R under conditions in which wild-type holoenzyme formed readily. Pairing rC(W196R) with mutant forms of R lacking domain B or having defects in cAMP binding sites A or B highlighted the importance of the conformation of R, and, in particular, the accessibility of site A. One of these mutants, rR(R333K), having a defect in cAMP binding site B formed a stable complex with rC(W196R) in the absence of cAMP. However, unlike wild-type holoenzyme, this complex was active.

Catalysis↗

PubMed1997

A stable alpha-helical domain at the N terminus of the RIalpha subunits of cAMP-dependent protein kinase is a novel dimerization/docking motif.

The RIalpha subunit of cAMP-dependent protein kinase is maintained as an asymmetric dimer by a dimerization motif at the N terminus. Based on resistance to proteolysis and expression as a discrete domain in Escherichia coli, this motif is defined as residues 12-61. This motif is chemically, kinetically, and thermally stable. The two endogenous interchain disulfide bonds between Cys16 and Cys37 in RIalpha are extremely resistant to reduction even in 8 M urea, indicating that they are well shielded from the reducing environment of the cell. The disulfide bonds were present in recombinant RIalpha as well as when the dimerization domain alone was expressed in E. coli, emphasizing the unusual stability of this motif and the disulfide bonds. Although 100 mM dithiothreitol was sufficient to reduce the disulfide bonds, it did not abolish dimerization. In addition, a stable dimer also still formed when Cys37 was replaced with His, confirming unambiguously the original antiparallel alignment of the disulfide bonds. Thus, both in vitro and in vivo, disulfide bonds are not required for dimerization. Circular dichroism of the dimerization domain indicated a high content of a thermostable alpha-helix. Based on the CD data, trypsin resistance of the fragment, location of the disulfide bonds, and amphipathic helix predictions, potential models are discussed. A new alignment of the dimerization domains of RI, RII, and cGMP-dependent protein kinase elucidates fundamental similarities as well as significant differences among these three domains.

Amino Acid Sequence↗

PubMed1997

D-AKAP2, a novel protein kinase A anchoring protein with a putative RGS domain.

Subcellular localization directed by specific A kinase anchoring proteins (AKAPs) is a mechanism for compartmentalization of cAMP-dependent protein kinase (PKA). Using a two-hybrid screen, a novel AKAP was isolated. Because it interacts with both the type I and type II regulatory subunits, it was defined as a dual specific AKAP or D-AKAP1. Here we report the cloning and characterization of another novel cDNA isolated from that screen. This new member of the D-AKAP family, D-AKAP2, also binds both types of regulatory subunits. A message of 5 kb pairs was detected for D-AKAP2 in all embryonic stages and in all adult tissues tested. In brain, skeletal muscle, kidney, and testis, a 10-kb mRNA was identified. In testis, several small mRNAs were observed. Therefore, D-AKAP2 represents a novel family of proteins. cDNA cloning from a mouse testis library identified the full length D-AKAP2. It is composed of 372 amino acids which includes the R binding fragment, residues 333-372, at its C-terminus. Based on coprecipitation assays, the R binding domain interacts with the N-terminal dimerization domain of RIalpha and RIIalpha. A putative RGS domain was identified near the N-terminal region of D-AKAP2. The presence of this domain raises the intriguing possibility that D-AKAP2 may interact with a Galpha protein thus providing a link between the signaling machinery at the plasma membrane and the downstream kinase.

A Kinase Anchor Proteins↗

PubMed1997

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.

Adenosine↗

PubMed1997

Role of the glycine triad in the ATP-binding site of cAMP-dependent protein kinase.

A glycine-rich loop in the ATP-binding site is one of the most highly conserved sequence motifs in protein kinases. Each conserved glycine (Gly-50, Gly-52, and Gly-55) in the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) was replaced with Ser and/or Ala. Active mutant proteins were expressed in Escherichia coli, purified to apparent homogeneity, separated into phosphoisoforms, and characterized. Replacing Gly-55 had minimal effects on steady-state kinetic parameters, whereas replacement of either Gly-50 or Gly-52 had major effects on both Km and kcat values consistent with the prediction of the importance of the tip of the glycine-rich loop for catalysis. Substitution of Gly-50 caused a 5-8-fold reduction in Km (ATP), an 8-12-fold increase in Km (peptide), and a 3-5-fold drop in kcat. The Km (ATP) and Km (peptide) values of C(G52S) were increased 8- and 18-fold, respectively, and the kcat was decreased 6-fold. In contrast to catalytic efficiency, the ATPase rates of C(G50S) and C(G52S) were increased by more than an order of magnitude. The thermostability of each mutant was slightly increased. Unphosphorylated C(G52S) was characterized as well as several isoforms phosphorylated at a single site, Ser-338. All of these phosphorylation-defective mutants displayed a substantial decrease in both enzymatic activity and thermal stability that correlated with the missing phosphate at Thr-197. These results are correlated with the crystal structure, models of the respective mutant proteins, and conservation of the Glys within the protein kinase family.

Adenosine Triphosphatases↗

PubMed1997

Interaction of the regulatory and catalytic subunits of cAMP-dependent protein kinase. Electrostatic sites on the type Ialpha regulatory subunit.

Since a basic surface on the catalytic (C) subunit of cAMP-dependent protein kinase is important for binding to the regulatory (R) subunit, acidic residues in R were sought that might contribute to R-C interaction. Using differential labeling by a water-soluble carbodiimide (Buechler, T. A., and Taylor, S. S. (1990) Biochemistry 29, 1937-1943), seven specific carboxylates in RIalpha were identified that were protected from chemical modification in the holoenzyme; each was then replaced with Ala. Of these, rRI(E15A/E106A/D107A)), rRI(E105A), rRI(D140A), rRI(E143A), and rRI(D258A) all were defective in holoenzyme formation and define negative electrostatic surfaces on RIalpha. An additional conserved carboxylate, Glu101 in RIalpha and the equivalent, Glu99 in RIIalpha were mutated to Ala. Replacement of Glu101 had no effect while rRII(E99A) was very defective. RIalpha and RIIalpha thus differ in the molecular details of how they recognize C. Unlike wild-type RI, two additional mutants, rRI(D170A) and rRI(K242A), inhibited C-subunit stoichiometrically in the presence of cAMP and show increases in both on- and off-rates. Asp170, which contributes directly to the hydrogen bonding network in cAMP-binding site A, thus contributes also to holoenzyme stability.

Amino Acid Sequence↗

PubMed1997

Identification of a partially rate-determining step in the catalytic mechanism of cAMP-dependent protein kinase: a transient kinetic study using stopped-flow fluorescence spectroscopy.

The kinetics of nucleotide binding and phosphoryl group transfer were measured in the catalytic subunit of cAMP-dependent protein kinase using stopped-flow fluorescence spectroscopy and an acrylodan-labeled derivative of this enzyme, which we have previously shown to have kinetic properties similar to those for the wild-type enzyme (Lew et al., 1996). The fluorescence emission spectrum of this enzyme is quenched differentially by ATP and ADP so that both the binding of ligands and phosphoryl group transfer at the active site can be monitored selectively. The association and dissociation rate constants for both nucleotides were measured using two methods: relaxation and competition binding. The ratio of the observed dissociation and association rate constants by both methods are consistent with Kd measurements (25 microM) determined by equilibrium fluorescence quenching. The dissociation rate constant for ADP (100 s(-1)) is approximately 2.5-fold larger than k(cat) (39 s(-1)). A full viscosity effect was measured for k(cat), suggesting that a diffusive step or steps limit maximum turnover. Pre-steady-state kinetic transients are biphasic and were fitted to observed rate constants of 500 s(-1) and 60 s(-1) at 500 microM Kemptide (LRRASLG). Metal substitution studies (Mg2+ vs Mn2+) indicate that this first phase represents the phosphoryl group transfer step. Phosphopeptide release is faster than this second phase since the substrate is in rapid exchange with the enzyme and phosphorylation reduces the affinity of the peptide. The inability to assign this second phase to the chemical event or to product release implies that it reflects a viscosity-sensitive, protein conformational change that occurs after phosphoryl group transfer and prior to product release. Two conformational steps were detected in the binding of both ATP and ADP by relaxation methods that may be related to this second pre-steady-state kinetic phase. We suggest that this additional step in the kinetic mechanism may also occur in the wild-type enzyme and represents a large structural change in the enzyme during normal catalytic cycling.

Adenosine Diphosphate↗

PubMed1997

Kinetochore localization of murine Bub1 is required for normal mitotic timing and checkpoint response to spindle damage.

The mitotic checkpoint ensures proper chromosome segregation by delaying anaphase until chromosomes are aligned on the spindle. Following prolonged spindle damage, however, cells eventually exit mitosis and undergo apoptosis. We show here that a murine homolog of the yeast mitotic checkpoint gene BUB1 localizes to the kinetochore during mitosis. By expressing a dominant-negative mutant, we show that mBub1 is not only required for the checkpoint response to spindle damage, but acts in the timing of a normal mitosis. In addition, when mBub1 function is compromised, cells escape apoptosis and continue cell cycle progression, despite leaving mitosis with a disrupted spindle. These data demonstrate a role for kinetochore-associated mBub1 in regulating exit from mitosis, and suggest functional links between the mitotic checkpoint and subsequent apoptotic events in G1.

Amino Acid Sequence↗

PubMed1997

Crystal structure of a polyhistidine-tagged recombinant catalytic subunit of cAMP-dependent protein kinase complexed with the peptide inhibitor PKI(5-24) and adenosine.

The crystal structure of the hexahistidine-tagged mouse recombinant catalytic subunit (H6-rC) of cAMP-dependent protein kinase (cAPK), complexed with a 20-residue peptide inhibitor from the heat-stable protein kinase inhibitor PKI(5-24) and adenosine, was determined at 2.2 A resolution. Novel crystallization conditions were required to grow the ternary complex crystals. The structure was refined to a final crystallographic R-factor of 18.2% with good stereochemical parameters. The "active" enzyme adopts a "closed" conformation as found in rC:PKI(5-24) [Knighton et al. (1991a,b) Science 253, 407-414, 414-420] and packs in a similar manner with the peptide providing a major contact surface. This structure clearly defines the subsites of the unique nucleotide binding site found in the protein kinase family. The adenosine occupies a mostly hydrophobic pocket at the base of the cleft between the two lobes and is completely buried. The missing triphosphate moiety of ATP is filled with a water molecule (Wtr 415) which replaces the gamma-phosphate of ATP. The glycine-rich loop between beta1 and beta2 helps to anchor the phosphates while the ribose ring is buried beneath beta-strand 2. Another ordered water molecule (Wtr 375) is pentacoordinated with polar atoms from adenosine, Leu 49 in beta-strand 1, Glu 127 in the linker strand between the two lobes, Tyr 330, and a third water molecule, Wtr 359. The conserved nucleotide fold can be defined as a lid comprised of beta-strand 1, the glycine-rich loop, and beta-strand 2. The adenine ring is buried beneath beta-strand 1 and the linker strand (120-127) that joins the small and large lobes. The C-terminal tail containing Tyr 330, a segment that lies outside the conserved core, covers this fold and anchors it in a closed conformation. The main-chain atoms of the flexible glycine-rich loop (residues 50-55) in the ATP binding domain have a mean B-factor of 41.4 A2. This loop is quite mobile, in striking contrast to the other conserved loops that converge at the active site cleft. The catalytic loop (residues 166-171) and the Mg2+ positioning loop (residues 184-186) are a stable part of the large lobe and have low B-factors in all structures solved to date. The stability of the glycine-rich loop is highly dependent on the ligands that occupy the active site cleft with maximum stability achieved in the ternary complex containing Mg x ATP and the peptide inhibitor. In this ternary complex the gamma-phosphate is secured between both lobes by hydrogen bonds to the backbone amide of Ser 53 in the glycine-rich loop and the amino group of Lys 168 in the catalytic loop. In the adenosine ternary complex the water molecule replacing the gamma-phosphate hydrogen bonds between Lys 168 and Asp 166 and makes no contact with the small lobe. This glycine-rich loop is thus the most mobile component of the active site cleft, with the tip of the loop being highly sensitive to what occupies the gamma-subsite.

Adenine↗

PubMed1997

Identification of a novel protein kinase A anchoring protein that binds both type I and type II regulatory subunits.

Compartmentalization of cAMP-dependent protein kinase is achieved in part by interaction with A-kinase anchoring proteins (AKAPs). All of the anchoring proteins identified previously target the kinase by tethering the type II regulatory subunit. Here we report the cloning and characterization of a novel anchoring protein, D-AKAP1, that interacts with the N terminus of both type I and type II regulatory subunits. A novel cDNA encoding a 125-amino acid fragment of D-AKAP1 was isolated from a two-hybrid screen and shown to interact specifically with the type I regulatory subunit. Although a single message of 3.8 kilobase pairs was detected for D-AKAP1 in all embryonic stages and in most adult tissues, cDNA cloning revealed the possibility of at least four splice variants. All four isoforms contain a core of 526 amino acids, which includes the R binding fragment, and may be expressed in a tissue-specific manner. This core sequence was homologous to S-AKAP84, including a mitochondrial signal sequence near the amino terminus (Lin, R. Y., Moss, S. B., and Rubin, C. S. (1995) J. Biol. Chem. 270, 27804-27811). D-AKAP1 and the type I regulatory subunit appeared to have overlapping expression patterns in muscle and olfactory epithelium by in situ hybridization. These results raise a novel possibility that the type I regulatory subunit may be anchored via anchoring proteins.

A Kinase Anchor Proteins↗

PubMed1997

Recombinant strategies for rapid purification of catalytic subunits of cAMP-dependent protein kinase.

Knowledge of the crystal structure of the catalytic subunit (C) of cAMP-dependent protein kinase provided for the first time a molecular basis for probing function by site-directed mutagenesis. The purification of mutant C-subunits, however, presented new and unanticipated challenges due to instability, insolubility, and underphosphorylation of the altered proteins. To overcome these barriers, a rapid and efficient method for purifying recombinantly expressed C-subunits was developed. Purification to near homogeneity is achieved in less than 5 h. The procedure is based on colysis of bacteria that overexpress the C-subunit with bacteria that overexpress a poly-His-tagged mutant of the type II regulatory subunit H6RII (R213K). This mutant R-subunit with an altered cAMP binding site A forms holoenzyme rapidly in bacterial extracts, and the Ka (cAMP) for the resulting holoenzyme, 27-37 microM, is nearly 50-fold increased compared to holoenzyme formed with wild-type RII. Thus, after batchwise immobilizing the holoenzyme on Ni(2+)-resin, the free C-subunit can be directly eluted batchwise with high concentrations of cAMP. The method is described for the purification of wild-type C, with yields of approximately 5 mg/liter. In addition, a mutant subunit, C[G52S], which is defective in ATP binding and could not be isolated using previously described methods, was purified with equal efficiency.

Adenosine Triphosphate↗