Amino Acids. 2016 Dec;48(12):2875-2880. doi:10.1007/s00726-016-2345-6

Role of amino acid residues surrounding the phosphorylation site in peptide substrates of G protein-coupled receptor kinase 2 (GRK2)

Daisuke Asai,1 Hideki Nakashima,1 and Jeong-Hun Kang 2

1 Department of Microbiology St. Marianna University School of Medicine, Sugao 2-16-1 Miyamae, Kawasaki, 216-8511, Japan

2 Division of Biopharmaceutics and Pharmacokinetics, National Cerebral and Cardiovascular Center Research Institute, 5-7-1, Fujishiro-dai, Suita, Osaka 565-8565, Japan



We previously identified a β-tubulin-derived, 13-amino-acid peptide, 404DEMEFTEAESNMN416 (in which Thr-409 is the predominant phosphorylation site, while Ser-413 is weakly phosphorylated) as a specific substrate of G protein-coupled receptor kinase 2 (GRK2), and in this work, we made a series of amino acid substitutions in order to examine the influence of various residues on recognition by GRK2. Amino-terminal acetylation increased the affinity for GRK2. We also found that anionic amino acid residues around the phosphorylation site, e.g., at the -2 amino-terminal position and the +1 carboxyl-terminal position, are important for high affinity for GRK2. Finally, we discovered a modified peptide (Ac-EEMEFSEAEANMN-NH2, in which Ser is the phosphorylation site) that showed significantly higher affinity for GRK2 than the original peptide, with very high specificity for GRK2 over GRK5. This peptide may be a useful tool for investigating the diverse intracellular signaling pathways regulated by GRK2, and also for developing GRK2-specific inhibitors as candidate drugs, e.g., for treatment of cardiovascular diseases.




The G protein-coupled receptor kinases (GRKs) are cytosolic serine/threonine protein kinases and control signaling and expression of the G protein-coupled receptor (GPCR) family via phosphorylation of GPCRs. GRKs-mediated phosphorylation of GPCRs is associated with various diseases. Among GRKs, GRK2 (formerly known as β-adrenergic receptor kinase 1) is regarded as a therapeutic target for various diseases, including cancer, inflammation, and brain and cardiovascular diseases, because of its hyperactivation in these diseases and involvement in the control of disease-related signaling pathways [1]. It has been shown that selective inhibition of GRK2 activity is therapeutically effective and a valid strategy to treat those diseases [2]. GRK2ct is known as a peptide inhibitor specific for GRK2, and molecular information of peptide substrate-GRK2 interactions is often useful to design an effective inhibitor.


Development of peptide substrates of GRK2

We are interested in the application of substrate peptides to develop new tools, such as artificial sensors to detect hyper-activated enzymes in lysate of diseased cells [3]. GRK2-specific peptide substrates are expected to be useful as tools for investigating the diversity of intracellular signaling pathways regulated by GRK2 and also potentially for drug development. RESA peptide (RRREEEEESAAA) is the only small peptide so far reported as a substrate for GRK2, and has been used to investigate the phosphorylation reaction. This peptide was synthesized on the basis that peptide substrates of GRK2 require acidic residues to be localized on the N-terminal side of a Ser/Thr phosphate acceptor group. Several proteins have been reported as GRK2 phosphorylation targets, including β-tubulin, synucleins (α and β), Nedd4, Nedd4-2, β2-adrenergic receptor, epithelial Na+ channels, phosducin, rhodopsin, ribosomal protein P2, downstream regulatory element antagonist modulator (DREAM), p38 mitogen-activated protein kinase (MAPK), phosphodiesterase γ (PDEγ), platelet-derived growth factor receptor-β (PDGFR-β), Smad2, and ezrin/radixin. Although the patterns were not universal, consensus phosphorylation site motifs for GRK2 appeared to be (D/E)X1-3(S/T), (D/E)X1-3(S/T)(D/E), and (D/E)X0-2(D/E)(S/T) (Table 1). We used this information to design a series of peptide substrates in order to further define the substrate determinants for GRK2. Our design criteria for candidate substrate peptides based on putative consensus phosphorylation site motifs identified in GRK2 substrate proteins were: (1) the amino-acid sequences are taken directly from the sequences of the parent protein substrates without any change; (2) Ser/Thr is located in the center of the peptide as a phosphate acceptor group; (3) the peptide length is limited to 15 residues to allow easy chemical synthesis. We designed 17 peptides based on the above criteria, as shown in Table 1.


Table 1. Substrates for GRK2 and their phosphorylation sites 

All phosphorylation sites are indicated in bold italic type. A, alanine; C, cysteine; D, aspartate; E, glutamate; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; Y, tyrosine; V, valine; and W, tryptophan.



The designed peptides were synthesized and phosphorylated with recombinant GRK2. The incorporation of radioactivity [counts per minute (CPM)] for each peptide substrate in radiolabel assay using [γ-32P]ATP is presented in Figure 1. The peptide substrates GR-11-1 and GR-11-8, taken from β-tubulin and epithelial Na+ channel, respectively, showed higher CPM levels than the other peptides, and GR-11-1 was the most highly phosphorylated substrate [4]. It is noteworthy that GR-11-1 exhibited a four-times-higher CPM level than RESA peptide, which has been widely used as a synthetic peptide substrate for GRK2 (Km for RESA: 1.1 mM). These results were in good agreement with the phosphorylation ratios determined by MALDI-TOF MS analysis [5].




Figure 1. Results of radiolabel assay using [γ-32P]ATP for candidate substrate peptides. Known substrate peptide RRREEEEESAAA was used as a control. The kinase activity of recombinant GRK2 towards each peptide was determined by measuring 32P transfer from [γ-32P]ATP to each peptide according to the recommendations of the manufacturer. CPM: counts per minute; ATP, adenosine triphosphate.



Structural requirements of β-tubulin-derived peptide for phosphorylation with GRK2

In order to examine the influence of various residues on recognition by GRK2, we synthesized and assayed peptide analogues of the sequence surrounding the phosphorylation region. Three shorter peptides (GR-11-1-3, GR-11-1-4, and GR-11-1-5) showed significantly reduced phosphorylation, compared with parent GR-11-1, while extension at the amino-terminus of GR-11-1 had no significant effect (GR-11-1-1 and GR-11-1-2) (Figure 2). Interestingly, the effects of truncation of aspartic acid (D) at the -5 position or methionyl-asparagine (MN) at the +5 and +6 positions demonstrated that these residues are crucial. These results indicate that at least the 13-amino acid sequence of β-tubulin corresponding to GR-11-1 is essential for phosphorylation by GRK2. The CPM of GR-11-1 with a free amino group at the amino terminus was significantly lower, compared with the amino-terminally acetylated peptide. This result indicates that a structural element, acetyl-aspartate, is key for eliciting a molecular contact or interaction with GRK2.



Figure 2. Sequences of β-tubulin-derived peptide substrates with different lengths and their CPM levels. Each peptide substrate was subjected to radiolabel assay using [γ-32P]ATP. A, alanine; D, aspartate; E, glutamate; F, phenylalanine; G, glycine; M, methionine; N, asparagine; S, serine; and T, threonine.



The candidate amino acids for substitution were selected based on the sequences of known GRK2 protein substrates. To investigate the role of amino acid residues surrounding the phosphorylation site for GRK2, the glutamate residue at the -2 position was substituted by a positive amino acid residue (lysine) or serine, and/or the glutamate residue at the +1 position was substituted by a positive amino acid residue (histidine). The substitution of lysine at the -2 position (e.g., GR-14-1) dramatically reduced the CPM, compared with the non-modified peptide substrate (GR-11-1). Furthermore, the substitution of histidine at the +1 position (e.g., GR-14-3) also reduced CPM (Fig. 2). These results strongly suggest that GRK2 requires negative amino acid residues (e.g., glutamate or aspartate) near the phosphorylation site.

To find more sensitive peptide substrates for GRK2, we used consensus phosphorylation site motifs described above and the accumulated molecular data reported previously to design modified peptide substrates (GR-14-6 ~ GR-14-10) (Fig. 3). When aspartate at the -5 position and threonine at the phosphorylation site were replaced with glutamate and serine, respectively, the sensitivity to GRK2 was increased, though not substantially (see GR-14-7). However, the GR-14-8 peptide substrate modified with glutamate, serine, and alanine at positions -5, 0, and +4, respectively, showed significantly increased CPM compared with the original peptide, GR-11-1. Exchange between alanine and glutamate at positions +2 and +3 did not permit efficient phosphorylation by either GRK2. On the other hand, the GR-14-6 peptide substrate modified with glutamate and alanine at the -5 and +4 positions, respectively, showed significantly reduced phosphorylation efficiency with GRK2, compared with GR-11-1. The valine variant of GR-14-6 at the +2 position (GR-14-10) behaved similarly to the parent GR-14-6. Finally, we identified the GR-14-8 peptide substrate (Ac-EEMEFSEAEANMN-NH2) as a sensitive and selective peptide substrate for GRK2.

The kinetic analysis of origin peptide GR-11-1 and modified peptide GR-14-8 revealed that the Km value of GR-11-1 was 33.9 μM and Vmax was 0.35 pmol min1 mg-1 and the Km and Vmax values of GR-14-8 for GRK2 were 54.0 μM and 1.0 pmol min-1 mg-1, respectively. For evaluation to inhibit GRK2-mediated phosphorylation of substrates specifically, we used two specific GRK2 inhibitors, methyl 5-[2-(5-nitro-2-furyl)vinyl]-2-furoate (EMD Millipore, Billerica, MA, USA) and 3-[(4-methyl-5-pyridin-4-yl-1,2,4-triazol-3-yl)methylamino]-N-[[2-(trifluoromethyl)phenyl]methyl]benzamide hydrochloride (Hello Bio, Bristol, UK). After these GRK2 inhibitor treatments, phosphorylation of β-tubulin-derived peptides were dose-dependently reduced (unpublished results by Asai, D. and Kang, J.H.). Further, the phosphorylation reactions showed very low CPM levels when these β-tubulin-derived peptides were phosphorylated by GRK5 and/or other recombinant kinases, including PKA, PKCa, ROCK2, c-Src kinase, and IKKb.


Concluding remarks and perspectives

GRK2 specifically phosphorylates target proteins regulating downstream signals through GPCRs, and peptide substrates with high affinity and specificity are expected to be useful research tools and candidate drugs for cardiovascular and other diseases. In the present work, we identified peptide GR-11-1 (Ac-DEMEFTEAESNMN-NH2) and GR-14-8 (Ac-EEMEFSEAEANMN-NH2) as highly phosphorylated substrates of GRK2. In this regard, it is superior to the widely used substrate peptide RESA. We are currently examining the possibility that these peptides might be useful as a molecular probe for cardiovascular diagnosis.



Figure 3. Phosphorylation of modified peptide substrates by GRK2, shown in terms of radiolabel incorporation levels (CPM). Each peptide substrate was subjected to GRK2-mediated [γ-32P]ATP radiolabel incorporation assay. Peptide sequences are shown (the phosphorylation site is indicated by bold italic font). A, alanine; D, aspartate; E, glutamate; F, phenylalanine; H, histidine; K, lysine; M, methionine; N, asparagine; S, serine; and T, threonine; and V, valine.




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This work was supported by a Grant-in-Aid for Challenging Exploratory Research (KAKENHI Grant Number 15K12531 for J.H.K) and for Scientific Research (C) (15K01319 for H.N; 26460050 and 17K08254 for D.A.) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.