A potent and highly selective peptide substrate for protein kinase C assay

Toomik, Reet; Ek, Pia

doi:10.1042/bj3220455

Cited by 22 publications

(21 citation statements)

References 26 publications

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“…The SPOT method has been used successfully to identify substrate sequence motifs for a number of protein kinases (27,28). In this study, we have expanded the practical utility of the SPOT method and demonstrated that peptide libraries specifically designed to screen for binding combinations lead to peptides that are potent inhibitors of cGPK.…”

Section: Discussionmentioning

confidence: 99%

Highly specific, membrane-permeant peptide blockers of cGMP-dependent protein kinase Iα inhibit NO-induced cerebral dilation

Dostmann

Taylor

Nickl

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

146

139

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Arrays of octameric peptide libraries on cellulose paper were screened by using 32 protein kinase inhibitor ͉ combinatorial libraries ͉ SPOT method ͉ membrane translocation signal ͉ smooth muscle T he cGMP-dependent protein kinases type I␣ and I␤ (cGPK) act directly downstream in the NO-mediated signaling pathway, controlling a variety of cellular responses, ranging from smooth muscle cell relaxation to neuronal synaptic plasticity (1, 2). The structural similarity of cGPK and its closest relative, the cAMP-dependent protein kinase (cAPK), has made it difficult to study cGPK pathways independent of those mediated by cAPK, primarily because of the lack of potent and selective cGPK inhibitors. Because recent studies have suggested that cAMP and cGMP are each able to cross-activate either cGPK or cAPK under physiological conditions, the specific role for cGPK within the NO͞cGMP-mediated signaling pathway remains obscure (for a review see ref. 1). However, recent advances have clearly identified specific intracellular targets for the cGPK isozymes I␣ and I␤ (3, 4). Also, inactivation of the genes for cGPK I␣͞I␤ and cGPK II showed that the cGPK isozymes regulate distinct cellular functions by pathways separate from those mediated by cAPK (5, 6).Attempts to identify cGPK-selective inhibitor peptides based on the autoinhibitory domain of the enzyme or in vivo substrates have been tedious at best, because of the lack of a well defined consensus sequence. Only a relative preference for basic residues surrounding the phosphate acceptor site has been established (7). Various synthetic peptides have been used to analyze the sequence requirements for cGPK substrates (8-11). Recently, we developed an iterative approach using phosphorylation of peptide libraries on cellulose paper to determine a priori the substrate specificity of cGPK versus cAPK. Consequently, we identified the cGPK substrate sequence TQAKRKKSLAM-FLR, in which the serine represents the phosphate-acceptor site (12, 13). Substitution of this serine by alanine yielded cGPK inhibitors with K i values of 7.5-22 M (13) and improved cGPK͞cAPK selectivity, as has been reported with other synthetic peptide derivatives (14,15). However, all cGPK peptide inhibitors known so far lack satisfactory potency and selectivity.Here we report a peptide library screen specifically designed to select for tight binding peptides rather than substrate peptides. First, we took advantage of the autophosphorylation properties of cGPK, which provides the means to study the transient enzyme-peptide interactions. Second, we used peptide libraries that lack the phosphate acceptor residues serine and threonine to select for peptide binding over phosphorylation. Linking the best sequence from this screen to membrane translocation signals (MTS) for intracellular delivery resulted in the highly effective cGPK I␣ inhibitors DT-2 and DT-3. Finally, we have demonstrated that both peptides are powerful tools for studying the specific functional roles of cGPK in smooth muscle. MethodsEnzyme Prepa...

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Section: Discussionmentioning

confidence: 99%

Highly specific, membrane-permeant peptide blockers of cGMP-dependent protein kinase Iα inhibit NO-induced cerebral dilation

Dostmann

Taylor

Nickl

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

146

139

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“…3A). Inasmuch as both serine and threonine may serve as phosphorylation residues within the theoretical mutated PKC consensus sequence, these results suggest that phosphorylation may perhaps be an important regulator of ORNT1 function (13). Future studies will explore this intriguing observation.…”

mentioning

confidence: 96%

“…Furthermore, Thr 32 has been conserved throughout evolution thus suggesting an important role in ORNT1 protein function. Interestingly, this amino acid change, T32R, abolishes a theoretical protein kinase C (PKC) phosphorylation site (Ser/Thr-Xaa-Arg/Lys) (13).…”

Section: Missense Mutation (T32r) In Hhh Syndromementioning

confidence: 99%

Clinical and Functional Characterization of a Human ORNT1 Mutation (T32R) in the Hyperornithinemia-Hyperammonemia-Homocitrullinuria (HHH) Syndrome

et al. 2006

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ABSTRACT:We studied two related families (HHH013 and HHH015) with the hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome, a disorder of the urea cycle and ornithine degradation pathway, who have the same novel ornithine transporter (ORNT1) genotype (T32R) but a variable phenotype. Both HHH015 patients are doing well in school and are clinically stable; conversely, the three affected HHH013 siblings had academic difficulties and one suffered recurrent episodes of hyperammonemia and ultimately died. Overexpression studies revealed that the product of the ORNT1-T32R allele has residual function. Ornithine transport studies in HHH015 fibroblasts, however, showed basal activity similar to fibroblasts carrying nonfunctional ORNT1 alleles. We also examined two potential modifying factors, the ORNT2 gene and the mitochondrial DNA lineage (haplogroup). Haplogroups, associated with specific diseases, are hypothesized to influence mitochondrial function. Results demonstrated that both HHH015 patients are heterozygous for an ORNT2 gain of function polymorphism and belong to haplogroup A whereas the HHH013 siblings carry the wild-type ORNT2 and are haplogroup H. These observations suggest that the ORNT1 genotype cannot predict the phenotype of HHH patients. The reason for the phenotypic variability is unknown, but factors such as redundant transporters and mitochondrial lineage may contribute to the neuropathophysiology of HHH patients. T he HHH syndrome (OMIM #238970) is an autosomal recessive disorder of the urea cycle and ornithine degradation pathway caused by the deficient transport of ornithine across the inner mitochondrial membrane (1,2). The gene defective in HHH syndrome is the mitochondrial ornithine transporter (ORNT1) that is localized in the q14.1 region of Ch13 and is a member of the MCF of proteins that includes the uncoupling protein, carnitine/acyl-carnitine translocase, and the ADP/ATP transporter (3,4). The human ORNT1 gene is expressed in the periportal hepatocytes, which contain the urea cycle pathway, and in the pericentral hepatocytes and skin fibroblasts that express the ornithine degradation pathway (1,2). Physiologically, ORNT1 allows ornithine to serve as a substrate for the ornithine transcarbamylase (OTC) and ornithine amino transferase (OAT) reactions that produce citrulline and the two amino acids, glutamate and proline. In vitro studies have demonstrated that ORNT1 transports ornithine, lysine, and arginine across the inner mitochondrial membrane in exchange for a hydrogen ion and citrulline (5).Biochemically, HHH syndrome is characterized by persistent elevation of plasma ornithine, episodic or postprandial hyperammonemia, and the urinary excretion of homocitrulline and orotic acid (1). The homocitrulline is believed to be the product of transcarbamoylation of lysine whereas the orotic aciduria occurs secondary to decreased OTC activity. Numerous studies have clearly demonstrated a link between hyperammonemia and CNS pathology in urea cycle disorders. However, little is known ...

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“…The phosphorylatable serine residue of sucrose synthase 2 is surrounded by the peptide sequence RVLSRLHS 15 VRER, with several positively charged amino acids before and after the Ser15 residue. This means that the substrate specificity of the relevant plant protein kinase(s) should follow similar principles to that of a large group of mammalian protein kinases, showing selectivity for positively charged amino acids [8].…”

Section: Camentioning

confidence: 99%

“…The phosphorylation reactions were carried out at 30 8C essentially as described in [15]. The specific radioactivity of [g-32 P]ATP was 50±150 c.p.m.´pmol 21 .…”

Section: Phosphorylation Of Spots Peptidesmentioning

confidence: 99%

Peptide phosphorylation by calcium‐dependent protein kinase from maize seedlings

Loog

Toomik

Sak

et al. 2000

European Journal of Biochemistry

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Ca2+ -dependent protein kinase (CDPK-1) was purified from maize seedlings, and its substrate specificity studied using a set of synthetic peptides derived from the phosphorylatable sequence RVLSRLHS 15 VRER of maize sucrose synthase 2. The decapeptide LARLHSVRER was found to be efficiently phosphorylated as a minimal substrate. The same set of peptides were found to be phosphorylated by mammalian protein kinase Cb (PKC), but showed low reactivity with protein kinase A (PKA). Proceeding from the sequence LARLHSVRER, a series of cellulose-membrane-attached peptides of systematically modified structure was synthesised. These peptides had hydrophobic (Ala, Leu) and ionic (Arg, Glu) amino acids substituted in each position. The phosphorylation of these substrates by CDPK-1 was measured and the substrate specificity of the maize protein kinase characterised by the consensus sequence motif A/L -5 X -4 R -3 X -2 X -1 SX +1 R +2 Z +3 R +4 , where X denotes a position with no strict amino acid requirements and Z a position strictly not tolerating arginine compared with the other three varied amino acids. This motif had a characteristic sequence element RZR at positions +2 to +4 and closely resembled the primary structure of the sucrose synthase phosphorylation site. The sequence surrounding the phosphorylatable serine in this consensus motif was similar to the analogous sequence K/RXXS/TXK/R proposed for mammalian PKC, but different from the consensus motif RRXS/TX for PKA. Recently it has been shown that one of the key enzymes of sucrose metabolism in maize, sucrose synthase type 2, can be phosphorylated at Ser15 in vivo [6,7]. This reaction seems to have regulatory significance, as the catalytic properties of the phosphorylated enzyme were affected [6].The phosphorylatable serine residue of sucrose synthase 2 is surrounded by the peptide sequence RVLSRLHS 15 VRER, with several positively charged amino acids before and after the Ser15 residue. This means that the substrate specificity of the relevant plant protein kinase(s) should follow similar principles to that of a large group of mammalian protein kinases, showing selectivity for positively charged amino acids [8]. The most extensively investigated members of this group are PKC and protein kinase A (PKA).In the present work, the substrate specificity of a protein kinase purified from maize seedlings was investigated using synthetic peptides derived from the maize sucrose synthase 2 phosphorylation site sequence, which were phosphorylated by this kinase in a Ca 2+ -dependent manner. The influence of peptide length on phosphorylation effectiveness was studied, and the sequence LARLHSVRER was found to be the minimal peptide substrate for this enzyme. Using this sequence, a series of structurally varied analogues were synthesized as cellulosemembrane-attached (SPOTs TM membranes) peptides. In these peptides, all the positions were substituted with hydrophobic (Leu, Ala) and charged (Arg, Glu) amino acids. By systematically varying the peptide structure, we were able to o...

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A potent and highly selective peptide substrate for protein kinase C assay

Cited by 22 publications

References 26 publications

Highly specific, membrane-permeant peptide blockers of cGMP-dependent protein kinase Iα inhibit NO-induced cerebral dilation

Highly specific, membrane-permeant peptide blockers of cGMP-dependent protein kinase Iα inhibit NO-induced cerebral dilation

Clinical and Functional Characterization of a Human ORNT1 Mutation (T32R) in the Hyperornithinemia-Hyperammonemia-Homocitrullinuria (HHH) Syndrome

Peptide phosphorylation by calcium‐dependent protein kinase from maize seedlings

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