Cell-Penetrating Peptide Based on Myosin Phosphatase Target Subunit Sequence Mediates Myosin Phosphatase Activity
Myosin phosphatase (MP) holoenzyme consists of protein phosphatase-1 (PP1) catalytic subunit (PP1c) associated with myosin phosphatase target subunit-1 (MYPT1) and it plays an important role in mediating the phosphorylation of the 20 kDa light chain (MLC20) of myosin, thereby regulating cell contrac...
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| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2025-05-01
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| Series: | Biomolecules |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2218-273X/15/5/705 |
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| Summary: | Myosin phosphatase (MP) holoenzyme consists of protein phosphatase-1 (PP1) catalytic subunit (PP1c) associated with myosin phosphatase target subunit-1 (MYPT1) and it plays an important role in mediating the phosphorylation of the 20 kDa light chain (MLC20) of myosin, thereby regulating cell contractility. The association of MYPT1 with PP1c increases the phosphatase activity toward myosin; therefore, disrupting/dissociating this interaction may result in inhibition of the dephosphorylation of myosin. In this study, we probed how MYPT1<sup>32–58</sup> peptide including major PP1c interactive regions coupled with biotin and cell-penetrating TAT sequence (biotin-TAT-MYPT1) may influence MP activity. Biotin-TAT-MYPT1 inhibited the activity of MP holoenzyme and affinity chromatography as well as surface plasmon resonance (SPR) binding studies established its stable association with PP1c. Biotin-TAT-MYPT1 competed for binding to PP1c with immobilized GST-MYPT1 in SPR assays and it partially relieved PP1c inhibition by thiophosphorylated (on Thr696 and Thr853) MYPT1. Moreover, biotin-TAT-MYPT1 dissociated PP1c from immunoprecipitated PP1c-MYPT1 complex implying its holoenzyme disrupting ability. Biotin-TAT-MYPT1 penetrated into A7r5 smooth muscle cells localized in the cytoplasm and nucleus and exerted inhibition on MP with a parallel increase in MLC20 phosphorylation. Our results imply that the biotin-TAT-MYPT1 peptide may serve as a specific MP regulatory cell-penetrating peptide as well as possibly being applicable to further development for pharmacological interventions. |
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| ISSN: | 2218-273X |