Abstract
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.
Highlights
Phosphorylation by protein kinases plays an essential part in the signal transduction pathways that regulate cellular functions in response to extracellular signals
Five peptide substrates showing counts per minute (CPM) values of .35,000 for ROCK2 were in the order R134 (KSARKKRYTVVGNPYWM).R4 (RAKYKTLRQIR).R22 (KPARKKRYTVVGNPYWM).R133 (KSDRKKRYTVVGNPYWM).R135 (KADRKKRYTVVGNPYWM) (Fig. 2)
There have been many efforts to search for peptide substrates with high affinity and sensitivity to ROCK2
Summary
Phosphorylation by protein kinases plays an essential part in the signal transduction pathways that regulate cellular functions in response to extracellular signals It is a general mechanism for the control of intracellular processes [1]. 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]. ROCK could be a potential therapeutic target for tumors, neurological disorders and cardiovascular diseases
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