The cAMP-dependent protein kinase discriminates between prochiral hydroxyl groups

Kwon, Young Guen; Srinivasan, Jaya; Mendelow, Marianne; Pluskey, Scott; Lawrence, David S.

doi:10.1021/ja00069a072

Cited by 18 publications

(22 citation statements)

References 2 publications

(2 reference statements)

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“…At least for some substrates, the active site of conventional protein kinases does not seem to readily accommodate a methyl group on the ␤-carbon of the substrate side chain (36,37). Perhaps because of this bias, it is rare to find a conventional protein kinase that phosphorylates any one protein exclusively on threonine, much less a whole set of proteins.…”

Section: Fig 5 Acid Hydrolysis Time Coursementioning

confidence: 99%

Specific Phosphorylation of Threonine by theDictyostelium Myosin II Heavy Chain Kinase Family

Luo

Crawley

Steimle

et al. 2001

Journal of Biological Chemistry

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Dictyostelium myosin II heavy chain kinase A (MHCK A), MHCK B, and MHCK C contain a novel type of protein kinase catalytic domain that displays no sequence identity to the catalytic domain present in conventional serine, threonine, and/or tyrosine protein kinases. Several proteins, including myelin basic protein, myosin regulatory light chain, caldesmon, and casein were phosphorylated by the bacterially expressed MHCK A, MHCK B, and MHCK C catalytic domains. Phosphoamino acid analyses of the proteins showed that 91 to 99% of the phosphate was incorporated into threonine with the remainder into serine. Acceptor amino acid specificity was further examined using a synthetic peptide library (MAXXXX(S/T)XXXXAKKK; where X is any amino acid except cysteine, tryptophan, serine, and threonine and position 7 contains serine and threonine in a 1.7:1 ratio). Phosphorylation of the peptide library with the three MHCK catalytic domains resulted in 97 to 99% of the phosphate being incorporated into threonine, while phosphorylation with a conventional serine/threonine protein kinase, the p21-activated kinase, resulted in 80% of the phosphate being incorporated into serine. The acceptor amino acid specificity of MHCK A was tested directly by substituting serine for threonine in a synthetic peptide and a glutathione S-transferase fusion peptide substrate. The serine-containing substrates were phosphorylated at a 25-fold lower rate than the threonine-containing substrates. The results indicate that the MHCKs are specific for the phosphorylation of threonine.

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Section: Fig 5 Acid Hydrolysis Time Coursementioning

confidence: 99%

Specific Phosphorylation of Threonine by theDictyostelium Myosin II Heavy Chain Kinase Family

Luo

Crawley

Steimle

et al. 2001

Journal of Biological Chemistry

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“…This difference has only a small effect on the maximal rate constant, £ rat (20 We have previously demonstrated that SPKs will catalyze the phosphorylation of a wide variety of synthetic alcohol-containing residues, including achiral species. 6 This observation is particularly germane to inhibitor design since it implies that these enzymes are relatively accommodating with respect to incorporating a multitude of structural motifs within their active site regions. In adition, we have found that the active site substrate specificity of SPKs is remarkably disimilar, even for those enzymes that are closely related.…”

Section: Scheme IImentioning

confidence: 99%

“…The following features concerning this activity are notable: First, although vAbl will phosphorylate peptides containing either D-or L-tyrosine, the latter is a significantly more efficient (65-fold) substrate than the former. Second, the D-tyrosine-containing peptide (23) is a 5-fold poorer substrate than the corresponding achiral species (6). Consequently, it is clear that the inverted configuration associated with D-tyrosine, in conjunction with the amide substituent, actively interferes with the ability of this species to be recognized as a substrate for v-Abl.…”

Section: C-src Phosphorylates a D-tyrosine Residue In An Active Simentioning

confidence: 99%

“…For comparative purposes, we also investigated the inhibitory activity of the tyramine-containing analog 25. This alcohol-bearing residue is phosphorylated by c-Src when attached to the C-terminal position of Arg-Arg-Arg-Arg-Arg-Leu-Glu-Glu-Leu-Leu- (6). However, since we attached tyramine to the tetrapeptide Glu-Glu-Leu-Leu-, any phosphorylation of the aromatic alcohol will be "invisible" to the phosphocellulose paper disc detection method.…”

Section: C-src Phosphorylates a D-tyrosine Residue In An Active Simentioning

confidence: 99%

See 1 more Smart Citation

Study of Inhibitors of Neu and Related Tyrosine-Specific Protein Kinases: Implications for the Treatment of Breast Cancer.

Edelman¹

1998

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“…The tetrapeptide-oxime resin was prepared according to a previously described Boc protocol using an Advanced ChemTech Act 90 peptide synthesizer (14).…”

Section: Preparation Of Glu(o-t-butyl)-glu(o-t-butyl)-leu-leu-oxime Rmentioning

confidence: 99%

Nonphosphorylatable Tyrosine Surrogates

Niu

Lawrence

1997

Journal of Biological Chemistry

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Tyrosine-specific protein kinases are known to utilize short synthetic tyrosine-containing peptides as substrates and, as a consequence, a number of inhibitory peptides have been prepared by replacing the tyrosine moiety in these peptides with a nonphosphorylatable phenylalanine residue. Unfortunately, the inhibitory efficacy of these phenylalanine-based peptides is often disappointing. These results demonstrate the need for nonphosphorylatable tyrosine surrogates that enhance enzyme affinity. As a consequence, we prepared nearly two dozen different phenethylamine derivatives, attached them to the C terminus of an active site-directed peptide (Glu-Glu-Leu-Leu), and examined their effectiveness as inhibitors of pp60 c-src . Three derivatives exhibit enhanced inhibitory activity (relative to phenethylamine), including para-substituted sulfonamide and guanidino analogs as well as a pentafluoro-containing species. The para-sulfonamide derivative was selected for further study and was found to function as a competitive inhibitor versus variable peptide substrate and as a noncompetitive inhibitor versus variable ATP. In short, the enhanced inhibitory activity of the sulfonamide derivative is not due to the association of this moiety with the ATP binding site. Furthermore, peptides containing the para-guanidino and pentafluoro derivatives of phenylalanine were prepared. These species also display enhanced inhibitory activity toward pp60 c-src relative to the corresponding phenylalaninebased peptide.More than 2 decades ago, the cAMP-dependent protein kinase was shown to phosphorylate short synthetic peptides containing sequences that correspond to phosphorylated sites in intact proteins (1-4). This key observation, which has been demonstrated innumerable times for other protein kinases in the intervening years, has had a profound impact on the ability to examine the chemistry and biochemistry of this important family of enzymes. Synthetic peptides are readily available and, unlike common protein kinase substrates such as histones, can be prepared containing only a single site of phosphorylation. As a consequence, peptidic substrates have proven to be an indispensable tool in the many detailed enzymological studies that have been described for protein kinases.In addition to their clear enzymological utility, peptide-based substrates immediately suggest the likelihood that structurally analogous inhibitors can be prepared. Indeed, the incorporation of an alanine residue, in place of the phosphorylatable serine moiety in a cAMP-dependent protein kinase-directed peptide, generates a species that serves as a competitive inhibitor versus both peptide and protein substrates (4). Feramisco and Krebs (5) subsequently introduced other residues in place of serine, including glycine, valine, aspartic acid, and asparagine. However, none of these substitutions provides an inhibitor as powerful as the alanine-based peptide. Interestingly, a naturally occurring inhibitor of the cAMP-dependent protein kinase also contains an alanine res...

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The cAMP-dependent protein kinase discriminates between prochiral hydroxyl groups

Cited by 18 publications

References 2 publications

Specific Phosphorylation of Threonine by theDictyostelium Myosin II Heavy Chain Kinase Family

Specific Phosphorylation of Threonine by theDictyostelium Myosin II Heavy Chain Kinase Family

Study of Inhibitors of Neu and Related Tyrosine-Specific Protein Kinases: Implications for the Treatment of Breast Cancer.

Nonphosphorylatable Tyrosine Surrogates

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