Transduction of the N-terminal fragments of MYPT1 enhances myofilament Ca2+ sensitivity in an intact coronary artery.
OBJECTIVE
The region of the 110 kDa regulatory subunit (MYPT1) of smooth muscle myosin phosphatase involved in the regulation of contraction was determined under physiological conditions.


RESULTS
Using HIV Tat protein-mediated protein transduction, the N-terminal fragments of MYPT1 were introduced to the intact porcine coronary arterial strips. Pre-incubation with 3 micromol/L TAT-MYPT1(1-374), a construct containing the Tat peptide and the residues 1 to 374 of MYPT1, for 15 minutes augmented (2.4-fold) the subsequent contraction induced by adding 1.25 mmol/L of extracellular Ca2+ under 118 mmol/L K+ depolarization, with no augmentation of the [Ca2+]i elevation. The deletion of the Tat peptide, MYPT1(1-374), abolished the augmenting effect. TAT-MYPT1(1-296) demonstrated a weaker but significant augmentation (1.7-fold). However, TAT-MYPT1(1-171), TAT-MYPT1(39-374), TAT-MYPT1(39-296), and TAT-MYPT1(297-374) had no augmenting activity. The myosin light chain phosphorylation level as a function of extracellular Ca2+ concentrations was shifted to the left in the strips pretreated with TAT-MYPT1(1-374) compared with the control.


CONCLUSIONS
Region 1 to 296 was the minimal region involved in the enhancement of contraction, and region 297 to 374 played a supplemental role. These results suggested that the interaction mainly between catalytic subunit and MYPT1 play a critical role in the regulation of the endogenous myosin phosphatase in intact smooth muscle.
Hirano
Katsuya
K
Division of Molecular Cardiology, Research Institute of Angiocardiology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Derkach
Dmitry N
DN
Hirano
Mayumi
M
Nishimura
Junji
J
Takahashi
Shosuke
S
Kanaide
Hideo
H
D008841:Actin Cytoskeleton / Q000187:drug effects*; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D020134:Catalytic Domain; D002645:Chickens; D003331:Coronary Vessels / Q000187:drug effects* / Q000201:enzymology; D015696:Gene Products, tat / Q000502:physiology; D066298:In Vitro Techniques; D009131:Muscle, Smooth, Vascular / Q000201:enzymology; D009200:Myocardial Contraction; D043263:Myosin-Light-Chain Phosphatase / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D010446:Peptide Fragments / Q000494:pharmacology; D010766:Phosphorylation; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000502:physiology; D034421:Sus scrofa; D014161:Transduction, Genetic; D014661:Vasoconstriction / Q000502:physiology
D015696:Gene Products, tat; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D043263:Myosin-Light-Chain Phosphatase; D002118:Calcium