Molecular Mechanism for the Regulation of Rho-Kinase by Dimerization and Its Inhibition by Fasudil

SummaryRho-kinase is a key regulator of cytoskeletal events and a promising drug target in the treatment of vascular diseases and neurological disorders. Unlike other protein kinases, Rho-kinase requires both N- and C-terminal extension segments outside the kinase domain for activity, although the details of this requirement have been elusive. The crystal structure of an active Rho-kinase fragment containing the kinase domain and both the extensions revealed a head-to-head homodimer through the N-terminal extension forming a helix bundle that structurally integrates the C-terminal extension. This structural organization enables binding of the C-terminal hydrophobic motif to the N-terminal lobe, which defines the correct disposition of helix αC that is important for the catalytic activity. The bound inhibitor fasudil significantly alters the conformation and, consequently, the mode of interaction with the catalytic cleft that contains local structural changes. Thus, both kinase and drug conformational pliability and stability confer selectivity

A Proteomic Approach for Comprehensively Screening Substrates of Protein Kinases Such as Rho-Kinase

BACKGROUND: Protein kinases are major components of signal transduction pathways in multiple cellular processes. Kinases directly interact with and phosphorylate downstream substrates, thus modulating their functions. Despite the importance of identifying substrates in order to more fully understand the signaling network of respective kinases, efficient methods to search for substrates remain poorly explored. METHODOLOGY/PRINCIPAL FINDINGS: We combined mass spectrometry and affinity column chromatography of the catalytic domain of protein kinases to screen potential substrates. Using the active catalytic fragment of Rho-kinase/ROCK/ROK as the model bait, we obtained about 300 interacting proteins from the rat brain cytosol fraction, which included the proteins previously reported as Rho-kinase substrates. Several novel interacting proteins, including doublecortin, were phosphorylated by Rho-kinase both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity

KANPHOS: Kinase-associated neural phospho-signaling database for data-driven research

Protein phosphorylation, a key regulator of cellular processes, plays a central role in brain function and is implicated in neurological disorders. Information on protein phosphorylation is expected to be a clue for understanding various neuropsychiatric disorders and developing therapeutic strategies. Nonetheless, existing databases lack a specific focus on phosphorylation events in the brain, which are crucial for investigating the downstream pathway regulated by neurotransmitters. To overcome the gap, we have developed a web-based database named “Kinase-Associated Neural PHOspho-Signaling (KANPHOS).” This paper presents the design concept, detailed features, and a series of improvements for KANPHOS. KANPHOS is designed to support data-driven research by fulfilling three key objectives: (1) enabling the search for protein kinases and their substrates related to extracellular signals or diseases; (2) facilitating a consolidated search for information encompassing phosphorylated substrate genes, proteins, mutant mice, diseases, and more; and (3) offering integrated functionalities to support pathway and network analysis. KANPHOS is also equipped with API functionality to interact with external databases and analysis tools, enhancing its utility in data-driven investigations. Those key features represent a critical step toward unraveling the complex landscape of protein phosphorylation in the brain, with implications for elucidating the molecular mechanisms underlying neurological disorders. KANPHOS is freely accessible to all researchers at https://kanphos.jp

Balance between dopamine and adenosine signals regulates the PKA/Rap1 pathway in striatal medium spiny neurons

Medium spiny neurons (MSNs) expressing dopamine D1 receptor (D1R) or D2 receptor (D2R) are major components of the striatum. Stimulation of D1R activates protein kinase A (PKA) through Golf to increase neuronal activity, while D2R stimulation inhibits PKA through Gi. Adenosine A2A receptor (A2AR) coupled to Golf is highly expressed in D2R-MSNs within the striatum. However, how dopamine and adenosine co-operatively regulate PKA activity remains largely unknown. Here, we measured Rap1gap serine 563 phosphorylation to monitor PKA activity and examined dopamine and adenosine signals in MSNs. We found that a D1R agonist increased Rap1gap phosphorylation in striatal slices and in D1R-MSNs in vivo. A2AR agonist CGS21680 increased Rap1gap phosphorylation, and pretreatment with the D2R agonist quinpirole blocked this effect in striatal slices. D2R antagonist eticlopride increased Rap1gap phosphorylation in D2R-MSNs in vivo, and the effect of eticlopride was blocked by the pretreatment with the A2AR antagonist SCH58261. These results suggest that adenosine positively regulates PKA in D2R-MSNs through A2AR, while this effect is blocked by basal dopamine in vivo. Incorporating computational model analysis, we propose that the shift from D1R-MSNs to D2R-MSNs or vice versa appears to depend predominantly on a change in dopamine concentration

Identification of the Kinase-Substrate Recognition Interface between MYPT1 and Rho-Kinase

Protein kinases exert physiological functions through phosphorylating their specific substrates; however, the mode of kinase–substrate recognition is not fully understood. Rho-kinase is a Ser/Thr protein kinase that regulates cytoskeletal reorganization through phosphorylating myosin light chain (MLC) and the myosin phosphatase targeting subunit 1 (MYPT1) of MLC phosphatase (MLCP) and is involved in various diseases, due to its aberrant cellular contraction, morphology, and movement. Despite the importance of the prediction and identification of substrates and phosphorylation sites, understanding of the precise regularity in phosphorylation preference of Rho-kinase remains far from satisfactory. Here we analyzed the Rho-kinase–MYPT1 interaction, to understand the mode of Rho-kinase substrate recognition and found that the three short regions of MYPT1 close to phosphorylation sites (referred to as docking motifs (DMs); DM1 (DLQEAEKTIGRS), DM2 (KSQPKSIRERRRPR), and DM3 (RKARSRQAR)) are important for interactions with Rho-kinase. The phosphorylation levels of MYPT1 without DMs were reduced, and the effects were limited to the neighboring phosphorylation sites. We further demonstrated that the combination of pseudosubstrate (PS) and DM of MYPT1 (PS1 + DM3 and PS2 + DM2) serves as a potent inhibitor of Rho-kinase. The present information will be useful in identifying new substrates and developing selective Rho-kinase inhibitors