Synthetic hexapeptide substrates and inhibitors of 3':5'-cyclic AMP-dependent protein kinase.

Kemp, Bruce E.; Benjamini, E.; Krebs, Edwin G.

doi:10.1073/pnas.73.4.1038

Cited by 218 publications

(90 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Actually, type II R-subunit can be phosphorylated on the hinge region [2]. However, the substrate site in the R-subunit is unlikely to be the sole region of the interaction, because (1) the highest affinity of known peptide substrates for the C-subunit is in the micromolar range, whereas that of the R-subnit is in the subnanomolar range [3][4][5], and (2) a mutant R-subunit, in which two arginines in the substrate consensus sequence are replaced by alanines, has been found to form a holoenzyme that can be dissociated by cAMP, although it is no longer a substrate for the C-subunit [6]. These reports suggest the presence of an additional interaction(s) between the R-and C-subunits which is distinct from that through the substrate site of the R-subunit.…”

Section: Introductionmentioning

confidence: 99%

Biochemical evidence for the interaction of regulatory subunit of cAMP‐dependent protein kinase with IDA (Inter‐DFG‐APE) region of catalytic subunit

et al. 1996

View full text Add to dashboard Cite

To explore the structural basis required for the hoioenzyme formation of cAMP-dependent protein kinase, we have prepared rabbit anti-peptide antibodies that can block the holoenzyme formation without affecting the catalytic activity of the enzyme. The antibodies were raised against a specific site in the catalytic (C)-subunit, termed IDA (Inter-DFG-APE) region, which lies between the kinase subdomains VII and VIII. Although the C-subunit immunoprecipitated with anti-IDA antibodies could not form a stable complex with regulatory (R)-subunit, it was still susceptible to inhibition by the R-subunit or by PKI, a specific inhibitor peptide containing a pseudosubstrate site. These results indicate that there exists an IDA regionmediated interaction between the R-and C-subunits, which is distinct from that mediated through the substrate site and substrate binding site. In accordance with this idea, association of synthetic IDA peptides with the R-subunit was directly demonstrated by resonance mirror analysis. The calculated association constants of IDA peptides were high enough to suggest a possible involvement of the IDA region in the initial step of holoenzyme formation.

show abstract

Section: Introductionmentioning

confidence: 99%

Biochemical evidence for the interaction of regulatory subunit of cAMP‐dependent protein kinase with IDA (Inter‐DFG‐APE) region of catalytic subunit

et al. 1996

View full text Add to dashboard Cite

show abstract

“…Although much is known about the protein kinase family on the structural and functional levels (3)(4)(5)(6), less is known about the interactions of these enzymes with their physiological protein substrates. Protein kinases recognize and phosphorylate short peptide segments (consensus sequences) of 4 -10 residues based on the known phosphorylation sequences of their targets (7)(8)(9). Although these short segments (consensus sequences) are minimal substrates, they do not provide all the binding energy offered by the full-length protein substrate.…”

mentioning

confidence: 99%

Chemical Clamping Allows for Efficient Phosphorylation of the RNA Carrier Protein Npl3

Aubol

Ungs

Lukasiewicz³

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

Protein kinases phosphorylate the appropriate protein substrate by recognizing residues both proximal and distal to the site of phosphorylation. Although these distal contacts may provide excellent binding affinities (low K m values) through stabilization of the enzymesubstrate complex, these contacts could reduce catalytic turnover (decrease k cat ) through slow phosphoprotein release. To investigate how protein kinases can overcome this problem and maintain both high substrate affinities and high turnover rates, the phosphorylation of the yeast RNA transport protein Npl3 by its natural protein kinase, Sky1p, was evaluated. Sky1p bound and phosphorylated Npl3 with a K m that was 2 orders of magnitude lower than a short peptide mimic representing the phosphorylation site and only proximal determinants. Surprisingly, this extraordinary difference is not the result of high affinity Npl3 binding. Rather, Npl3 achieves a low K m through a rapid and favorable phosphoryl transfer step. This step serves as a chemical clamp that locks the protein substrate in the active site without unduly stabilizing the product phosphoprotein and slowing its release. The chemical clamping mechanism offers an efficient means whereby a protein kinase can simultaneously achieve both high turnover and good substrate binding properties.The phosphorylation of hydroxyl-containing amino acids (i.e. serine, threonine, and tyrosine) in eukaryotic proteins controls numerous physiological responses essential for cell function. The enzymes that catalyze this modification, the protein kinases, have been studied for their critical role in complex signaling cascades and for their potential as valuable chemotherapeutic targets in proliferative diseases (1, 2). Although much is known about the protein kinase family on the structural and functional levels (3-6), less is known about the interactions of these enzymes with their physiological protein substrates. Protein kinases recognize and phosphorylate short peptide segments (consensus sequences) of 4 -10 residues based on the known phosphorylation sequences of their targets (7-9). Although these short segments (consensus sequences) are minimal substrates, they do not provide all the binding energy offered by the full-length protein substrate. For instance, the catalytic efficiency of phosphorylating the protein substrate MAPKAP2 using p38 MAPK 1 is ϳ2 orders of magnitude higher than that for a 14-residue peptide based on the consensus sequence (10). In another example, Csk phosphorylates the protein substrate Lck Ͼ3 orders of magnitude more efficiently than a 9-residue peptide based on the phosphorylation site (11). Studies of this type support a model in which regions beyond the limited consensus sequence interact with the protein kinase scaffold and govern high efficiency protein phosphorylation.Although an x-ray structure for a protein kinase with a full-length protein substrate bound is currently unavailable, structural and functional studies are now beginning to reveal the nature of the interactin...

show abstract

“…As ADPribosyltransferase catalyzes the transfer of an ADP-ribose moiety from NAD to the arginine residue of acceptor proteins [6] and because this arginine is on the NHa-terminal side of the phosphoserine in almost all of the CAMPdependent protein kinase phosphorylation sites in which arginine is responsible for the enhanced phosphorylation of acceptor proteins [15,16], the possible regulation of the phosphoryIation of histones by ADP-ribosylation has to be considered.…”

Section: Resultsmentioning

confidence: 99%

ADP‐ribosylation regulates the phosphorylation of histones by the catalytic subunit of cyclic AMP‐dependent protein kinase

et al. 1983

View full text Add to dashboard Cite

Phosphorylation of whole histones from calf thymus by the catalytic subunit of cyclic AMP-dependent protein kinase was markedly reduced when the histones were ADP-ribosylated. NAD, nicotinamide or free ADP-ribose molecule did not suppress the phosphorylation.Urea gel electrophoretic analyses of the phosphorylated histones which had already been ADP-ribosylated revealed that the suppression of phosphorylation occurred in both Hl and core histones. Therefore, the possibility that ADP-ribosylation may regulate the phosphorylation of histones phosphorylation in nuclei warrants further investigation. MonofADP-ribosyl)ationADP-ribosyltransferase Pho.sphoryiation NAD CAMP-dependent protein kinase Histone

show abstract

Synthetic hexapeptide substrates and inhibitors of 3':5'-cyclic AMP-dependent protein kinase.

Cited by 218 publications

References 16 publications

Biochemical evidence for the interaction of regulatory subunit of cAMP‐dependent protein kinase with IDA (Inter‐DFG‐APE) region of catalytic subunit

Biochemical evidence for the interaction of regulatory subunit of cAMP‐dependent protein kinase with IDA (Inter‐DFG‐APE) region of catalytic subunit

Chemical Clamping Allows for Efficient Phosphorylation of the RNA Carrier Protein Npl3

ADP‐ribosylation regulates the phosphorylation of histones by the catalytic subunit of cyclic AMP‐dependent protein kinase

Contact Info

Product

Resources

About