Functional Malleability of the Carboxyl-terminal Tail in Protein Kinase A

Chestukhin, Anton; Litovchick, Larisa; Schourov, Dmitry V.; Cox, Sarah; Taylor, Susan S.; Shaltiel, Shmuel

doi:10.1074/jbc.271.17.10175

Cited by 27 publications

(50 citation statements)

References 31 publications

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“…Thus in a closed conformation with bound adenosine, ATP, AMP-PNP, or ADP, the hydroxyl of Tyr 330 is involved with a bound water-mediated interaction with the nucleoside/nucleotide. The Tyr 330 hydroxyl also makes a hydrogen bond with the P-3 Arg of the peptide inhibitor (Chestukhin et al, 1996).…”

Section: Discussionmentioning

confidence: 99%

“…In a recent study Tyr 330 was mutated to Ala, Ser, and Phe (Chestukhin et al, 1996). The mutant where Tyr was replaced with Ala had a significantly elevated K m for ATP (from 40 to 230 µM) as well as for peptide substrate.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, the same hydroxyl of Tyr 330 interacts with the carbonyl of Leu 49 and the 2′-OH of the ribose of adenosine, via a water bridge. Mutational studies indicate that Tyr 330 is important for both peptide binding and catalysis (Chestukhin et al, 1996). The P-3 Arg donates a hydrogen bond to the carbonyl oxygen of Thr 51 in the glycine-rich loop between -strands 1 and 2.…”

Section: Conformation Of the Peptide Inhibitor Pki(5-24) And Its Intementioning

confidence: 99%

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Crystal Structure of a Polyhistidine-Tagged Recombinant Catalytic Subunit of cAMP-Dependent Protein Kinase Complexed with the Peptide Inhibitor PKI(5−24) and Adenosine

et al. 1997

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The crystal structure of the hexahistidine-tagged mouse recombinant catalytic subunit (H 6 -rC) of cAMP-dependent protein kinase (cAPK), complexed with a 20-residue peptide inhibitor from the heatstable protein kinase inhibitor PKI(5-24) and adenosine, was determined at 2.2 Å 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 γ-phosphate of ATP. The glycine-rich loop between 1 and 2 helps to anchor the phosphates while the ribose ring is buried beneath -strand 2. Another ordered water molecule (Wtr 375) is pentacoordinated with polar atoms from adenosine, Leu 49 in -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 -strand 1, the glycine-rich loop, and -strand 2. The adenine ring is buried beneath -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 glycinerich loop (residues 50-55) in the ATP binding domain have a mean B-factor of 41.4 Å 2 . 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 Mg 2+ 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‚ATP and the peptide inhibitor. In this ternary complex the γ-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 γ-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 γ-subsite.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Conformation Of the Peptide Inhibitor Pki(5-24) And Its Intementioning

confidence: 99%

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Crystal Structure of a Polyhistidine-Tagged Recombinant Catalytic Subunit of cAMP-Dependent Protein Kinase Complexed with the Peptide Inhibitor PKI(5−24) and Adenosine

et al. 1997

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“…5 in Ref. 4), the loss of cleavability may result from the removal of a segment of the polypeptide backbone, which may well prevent amino acid residues following the deletion from reaching their counterparts in the core segment with which they interact in the wild-type enzyme structure. This kind of "shortened rope" effect may, of course, have a detrimental structural outcome in many deletion studies except when the deletion involves a loop that does not interact with the core of the protein.…”

Section: Figmentioning

confidence: 99%

Unveiling the Substrate Specificity of Meprin β on the Basis of the Site in Protein Kinase A Cleaved by the Kinase Splitting Membranal Proteinase

Chestukhin¹,

Litovchick²,

Muradov

et al. 1997

Journal of Biological Chemistry

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The kinase splitting membranal proteinase (KSMP) is a metalloendopeptidase that inactivates the catalytic (C) subunit of protein kinase A (PKA) by clipping off its carboxyl terminal tail. Here we show that this cleavage occurs at Glu 332 -Glu 333 , within the cluster of acidic amino acids (Asp 328 -Glu 334 ) of the kinase. The K m values of KSMP and of meprin ␤ (which reproduces KSMP activity) for the C-subunit are below 1 M. The K m for peptides containing a stretch of four Glu residues are in the micromolar range, illustrating the significant contribution of this cluster to the substrate recognition of meprin ␤. This conclusion is supported by a systematic study using a series of the C-subunit mutants with deletions and mutations in the cluster of acidics. Hydrophobic amino acids vicinal to the cleavage site increase the K cat of the proteinase. These studies unveil a new specificity for meprin ␤, suggesting new substrates that are 1-2 orders of magnitude better in their K m and K cat than those commonly used for meprin assay. A search for substrates having such a cluster of acidics and hydrophobics, which are accessible to meprin under physiological conditions, point at gastrin as a potential target. Indeed, meprin ␤ is shown to cleave gastrin at its cluster of five glutamic acid residues and also at the M-D bond within its WMDF-NH 2 sequence, which is indispensable for all the known biological activities of gastrins. The latter meprin cleavage will lead to the inactivation of gastrin and thus to the control of its activity.The presence of a kinase splitting membranal proteinase (KSMP) 1 in the brush-border membranes of the rat small intestine was demonstrated as early as 1979 (1). This proteinase was shown to clip the catalytic (C) subunit of PKA, yielding a distinct cleavage product (CЈ) that was found to be devoid of the kinase activity. The biochemical characterization of KSMP as a proteinase revealed that it is an intriguing enzyme with a combination of the following unique features. (a) KSMP cleaves the C-subunit when it is free but not when inhibited by its regulatory (R) subunits, as in the R 2 C 2 complex (1, 2). (b) This cleavage could not be simulated by other proteinases (trypsin, chymotrypsin, clostripain, and papain (2)), suggesting that its specificity is not due merely to an interdomain exposure in C. (c) The proteinase was found to single out and selectively cleave the C-subunit in the presence of the large number of other proteins found in crude extracts of different tissues (brain, liver, or muscle) (3). (d) KSMP was found to cleave the Csubunit in its native conformation but not if the kinase is pre-denatured (2). (e) It distinguishes between the "open" and "closed" conformations of the C-subunit (4) that were recently identified by x-ray crystallography of this kinase (5).The cleavage of the C-subunit by KSMP leads to the removal of the carboxyl terminus tail of this kinase and seemed to occur at a distinct site (6 -8). Interestingly, two other kinases, the EGF-and the insulin-receptor ki...

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“…This transition between open and closed conformations in PKA is governed, in part, by the binding of ATP and peptide or protein substrate; however, to have a fully active enzyme requires also specific interactions of the N-and C-tails with both lobes of the core. 8,9 Although the N-terminal regions of AGC kinases are important in unique ways for the function of the core, 10 they are not conserved throughout the family. In contrast, the C-tails are a conserved structural element of all AGC kinases, and the conformation of the C-tail fluctuates between static and dynamic states that correlate with opening and closing of the active site cleft.…”

Section: Introductionmentioning

confidence: 99%

A chimeric mechanism for polyvalent trans‐phosphorylation of PKA by PDK1

et al. 2009

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Phosphorylation on the activation loop of AGC kinases is typically mediated by PDK1. The precise mechanism for this in-trans phosphorylation is unknown; however, docking of a hydrophobic (HF) motif in the C-tail of the substrate kinase onto the N-lobe of PDK1 is likely an essential step. Using a peptide array of PKA to identify other PDK1-interacting sites, we discovered a second AGC-conserved motif in the C-tail that interacts with PDK1. Since this motif [FD(X) 1-2 Y/F] lies in the active site tether region and in PKA contributes to ATP binding, we call it the Adenosine binding (Ade) motif. The Ade motif is conserved as a PDK1-interacting site in Akt and PRK2, and we predict it will be a PDK1-interacting site for most AGC kinases. In PKA, the HF motif is only recognized when the turn motif Ser338 is phosphorylated, possibly serving as a phosphorylation ''switch'' that regulates how the Ade and HF motifs interact with PDK1. These results demonstrate that the extended AGC C-tail serves as a polyvalent element that transregulates PDK1 for catalysis. Modeling of the PKA C-tail onto PDK1 structure creates two chimeric sites; the ATP binding pocket, which is completed by the Ade motif, and the C-helix, which is positioned by the HF motif. Together, they demonstrate substrate-assisted catalysis involving two kinases that have co-evolved as symbiotic partners. The highly regulated turn motifs are the most variable part of the AGC C-tail. Elucidating the highly regulated cis and trans functions of the AGC tail is a significant future challenge.

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Functional Malleability of the Carboxyl-terminal Tail in Protein Kinase A

Cited by 27 publications

References 31 publications

Crystal Structure of a Polyhistidine-Tagged Recombinant Catalytic Subunit of cAMP-Dependent Protein Kinase Complexed with the Peptide Inhibitor PKI(5−24) and Adenosine

Crystal Structure of a Polyhistidine-Tagged Recombinant Catalytic Subunit of cAMP-Dependent Protein Kinase Complexed with the Peptide Inhibitor PKI(5−24) and Adenosine

Unveiling the Substrate Specificity of Meprin β on the Basis of the Site in Protein Kinase A Cleaved by the Kinase Splitting Membranal Proteinase

A chimeric mechanism for polyvalent trans‐phosphorylation of PKA by PDK1

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