Peptide Substrates for Rho-Associated Kinase 2 (Rho-Kinase 2/ROCK2)

Peptide substrates sensitive for a certain protein kinase could be important for new-drug development and to understand the mechanism of diseases. Rho-associated kinase (Rho-kinase/ROCK) is a serine/threonine kinase, and plays an important part in cardiovascular disease, migration and invasion of tumor cells, and in neurological disorders. The purpose of this study was to find substrates with high affinity and sensitivity for ROCK2. We synthesized 136 peptide substrates from protein substrates for ROCK2 with different lengths and charged peptides. Incorporation of 32P [counts per minute (CPM)] for each peptide substrate was determined by the radiolabel assay using [γ-32P]ATP. When the top five peptide substrates showing high CPMs (R4, R22, R133, R134, and R135) were phosphorylated by other enzymes (PKA, PKCα, and ERK1), R22, R133, and R135 displayed the highest CPM level for ROCK2 compared with other enzymes, whereas R4 and R134 showed similar CPM levels for ROCK2 and PKCα. We hypothesize that R22, R133, and R135 can be useful peptide substrates for ROCK2.


Introduction
Phosphorylation by protein kinases plays an essential part in the signal transduction pathways that regulate cellular functions in response to extracellular signals. It is also a general mechanism for the control of intracellular processes [1]. Designing specific peptide substrates for a certain protein kinase is indispensable for the characterization of (or search for) substrate proteins for enzymes, and is also important in explorations for new drugs. In general, peptide substrates specific to a certain protein kinase are identified using genetic variants of protein substrates and synthetic peptides [2][3][4].
Rho-associated kinase (Rho-kinase/ROCK; hereafter referred to as ROCK) is a serine/threonine kinase and plays an important part in various cellular functions. These include the contraction of smooth muscle, cell adhesion, and cytokinesis [5,6]. Overexpression of this enzyme has been associated with cardiovascular diseases such as hypertension and cerebral and coronary vasospasm [7][8][9]. Moreover, ROCK is closely associated with the migration and invasion of tumor cells [10,11]. Recent studies suggest that inhibition of ROCK not only increases cerebral blood flow and leads to protection against stroke, but enhances functional recovery in injured spinal cords [12][13][14]. Thus, ROCK could be a potential therapeutic target for tumors, neurological disorders and cardiovascular diseases.
The purpose of the present study was to find substrates with high affinity and sensitivity for ROCK2. We synthesized 136 peptide substrates from protein substrates for ROCK2 with different lengths and charged peptides. After determining the incorporation of radioactivity (counts per minute (CPM)) for each peptide substrate by the radiolabel assay using [c-32 P]ATP, kinetic properties for the final five top-ranked substrates were analyzed. We also examined the affinities of five substrates for other kinases (extracellular signalregulated kinase 1 (ERK1), protein kinase A (PKA), and protein kinase C (PKC)a) showing similar consensus sequences and interactive functions in intracellular signal transduction to ROCKs.

Results
To search for peptide substrates with high affinity and sensitivity for ROCK2, we synthesized 136 peptide substrates on the basis of the amino-acid sequences in ROCK protein substrates (Table S1  and Table S2). These peptide substrates showed a different number of charged peptide residues or amino-acid residues. Peptide substrates had a basic amino acid (Arg or Lys) at aminoterminal position 22 or 23 of the phosphorylated sites (Ser and Thr). Moreover, most amino acids at the +2 carboxyl-terminal position were basic or hydrophobic (Arg, Lys, Phe, Leu, Trp, or Val) (Table S2).
The Ser:Thr ratio for the total number of phosphorylation sites used in this study was 9:12. The substitution of Ser for Thr at the phosphorylation site led to a tendency to a decrease in the radioactivity (see R1-6 and R7-12, R64-67 and R68-71, R72-75 and R76-79, R80-82 and R83-85, and R86-88 and R89-91) ( Fig. 1). Although the observation was not universal, the change of negatively charged amino acids (Asp or Glu) into Ala or positively charged amino acids tended to result in an increase in CPM.
However, the length of peptides had no or very little effect on CPMs ( Fig. 1 and Table S2).
Furthermore, the kinetic properties of five peptide substrates showing high CPM for ROCK2 were analyzed. Values of K m and V max for five peptide substrates ranged from 1.7 mM to 3.3 mM, and from 8.6 pmol/min/mg to 14.9 pmol/min/mg, respectively ( Table 1).
Several studies have reported that replacement of phosphorylatable amino acids with alanine can lead to an inhibitor peptide for each enzyme [38,39]. Thus, we examined if peptides that replace phosphorylatable amino acids with alanine can inhibit the phosphorylation of substrate peptide by ROCK2. The phosphorylatable threonine of five peptide substrates (R4, R22, R133, R134, and R135) was replaced with alanine and used as inhibitor peptides. The lowest K i (11.6 mM) in a competitive manner was obtained for R22 with alanine instead of threonine (Table 2).

Discussion
There have been many efforts to search for peptide substrates with high affinity and sensitivity to ROCK2. However, different experimental methods make selecting substrates for ROCK2 quite difficult. Moreover, the affinity and sensitivity of substrates for ROCK2 can be influenced by several factors. These include the peptide length and the number of charged peptides [38,[40][41][42], but these factors were not investigated in previous studies.
We synthesized 136 peptide substrates for ROCK2 with different lengths and charged peptides, and analyzed their ability for phosphorylation. When negatively charged amino acids (Asp and Glu) were replaced by Ala or a positively charged amino acid (Arg), resulting in an increase in the positive charge density in peptide substrate, a tendency to an increase in the radioactivity was identified. For example, a higher CPM value was identified for R4 than for R1, R38 than R33, and R134 than R133. The length of peptide substrate had no or little effect on the level of radioactivity. In general, shortening of peptide substrates decreased the affinity for the enzyme (i.e., increased the K m value).
However, the peptide length showed minor changes in the V max value [38,40,41]. These data showed that radioactivity was not affected by the length of peptide substrates, but that the change of negatively charged amino acids into Ala or positively charged amino acids could increase the radioactivity values.
PKA and PKCa broadly recognize ROCK phosphorylation sites because of their similar consensus sequences to the ROCK phosphorylation motif. Our previous study showed that ROCK substrates can be good substrates for PKA and/or PKCa. were phosphorylated by PKA and/or PKCa [33]. Moreover, there was a close function in intracellular signal transduction between ROCK and PKCa, ERK1, or PKA. The interactive role of ROCK and PKCa has been reported in the actin-myosin interaction [43] and in the contraction of vascular smooth muscle [44,45]. MLC phosphorylation was stimulated by ROCK and   PKCa, but decreased by suppressing ROCK and PKCa using inhibitors [44,45]. RhoA phosphorylation at Ser 188 by PKA blocks the ROCK pathways, resulting in activation of MLC phosphatase and the relaxation of smooth muscle [46]. Activated PKA can also facilitate axon formation by inhibiting RhoA, but ROCK has opposite effects on axon formation [47]. ROCK has an effect on translocation of ERK1 to the cellular nucleus, leading to proliferation of smooth muscle cells in the pulmonary artery [48]. ERK1 activation by ROCK stimulates: 1) force-induced osteopontin expression in human periodontal ligament fibroblasts through focal adhesion kinase signaling [49]; and 2) migration and proliferation of glioblastoma cells [50].  (Fig. 3). These results suggested that R22 and R133 could be useful peptide substrates for ROCK2, and that R4 and R134 could be useful peptide substrates for ROCK2 and PKCa. Replacement of phosphorylation sites with alanine is used to develop inhibitor peptides. In general, peptides showing higher affinity (i.e., low K m ) than lower affinity (i.e., high K m ) become much more effective inhibitor peptides when phosphorylation sites are substituted by alanine. In the present study, we examined if five peptides in which a phosphorylatable amino acid (threonine) was replaced with alanine could be used as potent competitive inhibitors of ROCK2. The R22 peptide with alanine instead of threonine showed the lowest K i (11.6 mM) ( Table 2).
In conclusion, there has been an increasing interest in ROCKs as therapeutic targets of cardiovascular disease, migration and invasion of tumor cells, and neurological disorders. Peptide substrates with high affinity and sensitivity to ROCK2 are important in new-drug developments and in understanding the cellular signals involved. In the present study, 136 peptide substrates from protein substrates for ROCK2 were synthesized and the radioactivity of each peptide substrate analyzed. From CPM results and phosphorylation reactions with other enzymes (PKA, PKCa, and ERK1), we found three peptides (R22, R133, and R135) to be useful peptide substrates for ROCK2.

Syntheses of peptide substrates
Peptide substrates were synthesized using an automatic peptide synthesizer according to standard Fmoc-chemistry procedures. After treatment with trifluoroacetic acid (TFA), peptides were purified on an Inertsil ODS-3 column (250620 mm, 3.5 mm; GL Sciences, Tokyo, Japan) using a BioCAD Perfusion Chromatography System (Ikemoto Scientific Technology, Tokyo, Japan) and a linear A-B gradient at a flow rate of 8 mL/min where eluent A was 0.1% TFA in water and eluent B was 0.1% TFA in acetonitrile. The purity of synthetic peptide was identified by highperformance liquid chromatography and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, and the peptide with .95% purity was used for the phosphorylation reaction.
The  (25 mM Tris-HCl at pH 7.5, 20 mM EGTA, 15 mM magnesium acetate, and 100 mM ATP) containing peptides and 0.5 mg/mL ERK1. For each experimental condition, values for control reactions lacking substrate peptides were subtracted as blanks. The assay mixture was incubated for 10 min at 25uC, and the reaction terminated by the addition of 5 mL of 30% trichloroacetic acid (TCA). The reaction mixture (24 mL) was spotted onto P-81 phosphocellulose membranes. The membranes were washed thrice with 5% TCA, dried with acetone, and the radioactivity of each membrane determined by liquid scintillation counting. Kinetic analyses of enzyme activity were determined using the Lineweaver-Burk plot.

Inhibition of phosphorylation by inhibitor peptides
The phosphorylatable amino acid (threonine) of five peptide substrates (R4, R22, R133, R134, and R135) was replaced with alanine. Inhibitory activity of these inhibitor peptides against ROCK2 was examined using S6K peptide (KRRRLASLR) as a substrate; value of K m and V max for S6K was 3.1 mM and 23.1 pmol/min/mg, respectively. K i values were obtained from the Dixon plot and Lineweaver-Burk plot. Inhibitor peptides were added into 25 mL of buffer (5 mM MOPS at pH 7.2, 2.5 mM beta-glycerophosphate, 1 mM EGTA, 4 mM MgCl 2 , 0.05 mM DTT, and 100 mM ATP) containing S6K substrate peptide and 1.25 mg/mL ROCK2. Supporting Information

Author Contributions
Performed the experiments: J-HK YK. Analyzed the data: J-HK DA AT. Contributed reagents/materials/analysis tools: DA TM TN. Wrote the paper: J-HK YK.