WB Recommended dilution: WB: 1 μg/mL Each laboratory should determine an optimum working titer for use in its particular application. Other applications have not been tested but use in such assays should not necessarily be excluded.
General Notes
This antibody was raised in sheep against MYPT1 (p-S445 aa 437-452). The non-phosphorylated peptides used to deplete nonphosphorylated specific Abs will be provided together. MYPT1 (Myosin Phosphatase Target Subunit 1) is a regulatory protein that plays a crucial role in the regulation of smooth muscle contraction by modulating myosin light chain phosphorylation.
Target
Alternative Names
PPP1R12A; protein phosphatase 1, regulatory subunit 12A; MBS; M130; MYPT1; protein phosphatase 1 regulatory subunit 12A; myosin binding subunit; myosin phosphatase, target subunit 1; myosin phosphatase-targeting subunit 1; protein phosphatase myosin-binding subunit; protein phosphatase 1, regulatory (inhibitor) subunit 12A;
Applications: WB Reactive species: Mouse
"Abstract: The induction of immunogenic cell death (ICD) impedes tumor progression via both tumor cell-intrinsic and -extrinsic mechanisms, representing a robust therapeutic strategy. However, ICD-targeted therapy remains to be explored and optimized. Through kinome-wide CRISPR-Cas9 screen, NUAK family SNF1-like kinase 1 (NUAK1) is identified as a potential target. The ICD-provoking effect of NUAK1 inhibition depends on the production of reactive oxygen species (ROS), consequent to the downregulation of nuclear factor erythroid 2-related factor 2 (NRF2)-mediated antioxidant gene expression. Moreover, the mevalonate pathway/cholesterol biosynthesis, activated by spliced form of X-box binding protein 1 (XBP1s) downstream of ICD-induced endoplasmic reticulum (ER) stress, functions as a negative feedback mechanism. Targeting the mevalonate pathway with CRISPR knockout or the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) inhibitor simvastatin amplifies NUAK1 inhibition-mediated ICD and antitumor activity, while cholesterol dampens ROS and ICD, and therefore also dampens tumor suppression. The combination of NUAK1 inhibitor and statin enhances the efficacy of anti-PD-1 therapy. Collectively, our study unveils the promise of blocking the mevalonate-cholesterol pathway in conjunction with ICD-targeted immunotherapy." Article snippet: Anti-MYPT1 (phospho S445 aa 437–452) polyclonal antibody, Creative Diagnostics Cat# CABT-BL6360; RRID:AB_3674399
Figure 1. Western blots of MYPT1 and its Ser445 phosphorylation in colorectal cancer cell lines upon vehicle and 10 mM or 20 mM HTH-01-015 treatments for 24 h.
Background
Three Myosin light chain phosphatase (MLCP) subunits are made: a catalytic subunit of type I protein phosphatase (PP1cδ), two non-catalytic subunits, myosin phosphatase target subunit 1 (MYPT1), and one subunit called M20. MYPT1 attaches to PP1cδ and M20, and sends PP1c to the phosphorylated sites in MLC. The complete enzyme complex containing MYPT1 effectively dephosphorylates the regulatory light chain of myosin II (MLC), thereby controlling actomyosin contraction in smooth muscle and non-muscle cells.
MYPT1, the regulatory subunit of MLCP, is a widely distributed polypeptide with a molecular weight of approximately 115kD. As a member of the MYPT family, it is highly expressed in smooth muscle tissue where it regulates muscle contraction. Beyond smooth muscle, MYPT1 is expressed in various cells and tissues, serving as an interaction platform for multiple proteins including Tau, MAP2, and GTP-RhoA, suggesting its diverse functional roles. The structure of MYPT1 includes several distinct domains: near the N-terminus, there is a binding site for the catalytic subunit PP1cδ and several ankyrin repeat sequences. The central region contains acidic and serine/threonine domains, while the C-terminal region features a leucine zipper (LZ) alternative splicing domain. The MYPT1 family undergoes alternative splicing primarily in the central insert (CI) exon and LZ exon, resulting in eight possible isoforms. The CI exon isoform switching in mammals is relatively conserved. While specific exon alternative splicing is conserved, the CI exon of MYPT1 can undergo alternative splicing in almost all species, though the functional significance of this regional variation remains unclear.
Figure 1. Domain structure of MYPT1 (Source: MacDonald JA, et al. 2018)
The regulation of MYPT1 function is associated with phosphorylation at T696 and T853 in the central region following the ankyrin repeat domain. Various protein kinases, including ROK, ZIPK, and ILK, can directly phosphorylate the T696 residue. ROK regulates MYPT1 through phosphorylation at both T696 and T853 sites. Phosphorylation at T696 directly reduces MLCP activity, while phosphorylation at T853 decreases MLCP activity by inducing the dissociation of MYPT1 from myosin. MYPT1 plays a crucial role in the GTPase Rho signaling pathway. Rho kinase increases smooth muscle contraction by depressing MYPT1 phosphorylation of myosin. They found that MYPT1 regulates cell migration, cell adhesion and cell cycle regulation. MYPT1 is localized near stress fibers and the cell membrane, where it participates in the regulation of cell morphology and movement.
1. MacDonald JA, et al. Regulation of Smooth Muscle Myosin Light Chain Phosphatase by Multisite Phosphorylation of the Myosin Targeting Subunit, MYPT1. Cardiovasc Hematol Disord Drug Targets. 2018;18(1):4-13.
2. Kiss A, et al. Myosin phosphatase: Unexpected functions of a long-known enzyme. Biochim Biophys Acta Mol Cell Res. 2019 Jan;1866(1):2-15.
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References
PPP1R12A Pathogenic Variants Associated with Human Congenital Malformations Syndrome
The transcriptional cofactor nascent polypeptide-associated complex and co-regulator (NACA) regulates osteoblast maturation and activity. NACA functions, at least in part, by binding to Jun proto-oncogene, AP-1 transcription factor subunit (cJUN) and potentiating the transactivation of AP-1 targets such as osteocalcin (Bglap) and matrix metallopeptidase 9 (Mmp9). NACA activity is modulated by phosphorylation carried out by several kinases, but a phosphatase regulating NACA's activity remains to be identified. Here, we used affinity purification with MS in HEK293T cells to isolate NACA complexes and identified protein phosphatase 1 catalytic subunit (PP1A) as a NACA-associated Ser/Thr phosphatase. NACA interacted with multiple components of the PP1A holoenzyme complex: the PPP1CA catalytic subunit and the regulatory subunits PPP1R9B, PPP1R12A and PPP1R18. MS analysis revealed that NACA co-expression with PPP1CA causes dephosphorylation of NACA at Thr-89, Ser-151, and Thr-174. NACA Ser/Thr-to-alanine variants displayed increased nuclear localization, and NACA dephosphorylation was associated with specific recruitment of novel NACA interactants, such as basic transcription factor 3 (BTF3) and its homolog BTF3L4. NACA and PP1A cooperatively potentiated cJUN transcriptional activity of the AP-1-responsive MMP9-luciferase reporter, which was abolished when Thr-89, Ser-151, or Thr-174 were substituted with phosphomimetic aspartate residues. We confirmed the NACA-PP1A interaction in MC3T3-E1 osteoblastic cells and observed that NACA phosphorylation status at PP1A-sensitive sites is important for the regulation of AP-1 pathway genes and for osteogenic differentiation and matrix mineralization. These results suggest that PP1A dephosphorylates NACA at specific residues, impacting cJUN transcriptional activity and osteoblast differentiation and function.
COA
Publication 
Figure 1. Domain structure of MYPT1
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