Role of Myosin Phosphatase Isoforms in cGMP-mediated Smooth Muscle Relaxation

Abstract

In vitro experiments showing the activation of the myosin phosphatase via heterophilic leucine zipper interactions between its targeting subunit (MYPT1) and cGMP-dependent protein kinase I suggested a pathway for smooth muscle relaxation (Surks, H. K., Mochizuki, N., Kasai, Y., Georgescu, S. P., Tang, K. M., Ito, M., Lincoln, T. M., and Mendelsohn, M. E. (1999) Science 286, 1583-1587). The relationship between MYPT1 isoform expression and smooth muscle responses to cGMP signaling in vivo has not been explored. MYPT1 isoforms that contain or lack a C-terminal leucine zipper are generated in birds and mammals by cassette-type alternative splicing of a 31-nucleotide exon. The avian and mammalian C-terminal isoforms are highly conserved and expressed in a tissue-specific fashion. In the mature chicken the tonic contracting aorta and phasic contracting gizzard exclusively express the leucine zipper positive and negative MYPT1 isoforms, respectively. Expression of the MYPT1 isoforms is also developmentally regulated in the gizzard, which switches from leucine zipper positive to negative isoforms around the time of hatching. This switch coincides with the development in the gizzard of a cGMP-resistant phenotype, i.e. inability to dephosphorylate myosin and relax in response to 8-bromo-cGMP after calcium activation. Furthermore, association of cGMP-dependent protein kinase I with MYPT1 is detected by immunoprecipitation only in the tissue that expresses the leucine zipper positive isoform of MYPT1. These results suggest that the regulated splicing of MYPT1 is an important determinant of smooth muscle phenotypic diversity and the variability in the response of smooth muscles to the calcium desensitizing effect of cGMP signaling.Smooth muscle contraction is initiated by the phosphorylation of the regulatory myosin light chain (MLC 20 ) 1 by the calcium/calmodulin-dependent activation of the myosin light chain kinase (MLCK) (1). Relaxation is effected by the dephosphorylation of MLC 20 by the smooth muscle myosin phosphatase (SMMP). Complexity is brought to this system by accessory proteins and signaling pathways that regulate the smooth muscle contractile state (reviewed in Refs. 2 and 3). The SMMP is a target of signals that are positive and negative modulators of smooth muscle tone. SMMP is a heterotrimeric protein composed of the 37-kDa catalytic subunit (PP1c␦), the 130/133-kDa myosin targeting subunit (MYPT1, also referred to as MBS), and the 21-kDa M21 subunit (4 -6). MYPT1 targets the catalytic subunit to MLC 20 (7,8) and in this way confers substrate specificity to the phosphatase, whereas the function of the M21 subunit is unknown. Activation of the Rho kinase signaling pathway leads to phosphorylation of the MYPT1 subunit, resulting in inhibition of myosin phosphatase activity and an increase in smooth muscle tone (9 -13). This signaling pathway is thought to determine the calcium-sensitizing effect of ␣-adrenergic stimulation, for example, in which greater force is produced at a given calcium concentration th...

Myosin Phosphatase-Rho Interacting Protein

“…M-RIP is thus localized to the contractile filament where myosin light chain phosphorylation regulates the contractile state, suggesting a role for M-RIP in myosin phosphatase regulation. The amino terminus of M-RIP contains adjacent pleckstrin homology domains and polyproline motifs, a structural combination also found on Bruton's tyrosine kinase where it mediates binding to both actin and G␣ 12 (50,51). This region of p116 RIP3 has recently been shown to mediate actin binding and actin bundling activity in vitro (49).…”

“…The carboxyl terminus of MBS contains a leucine zipper domain that mediates binding to other coiled-coil domains, including the leucine/isoleucine zipper domain of cGMP-dependent protein kinase 1␣ (11,12,43). To determine whether this domain of MBS mediates binding to the CC2 of M-RIP, wild-type COOH-terminal MBS (MBS LZ ) and mutant COOHterminal MBS in which all leucines in the leucine zipper domain were mutated to alanine (MBS LZ mutant), were expressed as GST fusion proteins and tested for binding to full-length M-RIP.…”

“…Both vasoconstrictor signaling pathways, which lead to inhibition of the phosphatase and cell contraction (reviewed in Ref. 10), and vasodilator signaling pathways, which lead to cell relaxation via activation of myosin phosphatase have been recently defined (11)(12)(13)(14)(15).…”

ROS-mediated downregulation of MYPT1 in smooth muscle cells: a potential mechanism for the aberrant contractility in atherosclerosis

“…In the present study, our results showed that peroxynitrite treatment induced MYPT1 phosphorylation at both Thr696 and Thr850 in Modulation of MYPT1 abundance and differential expression of spliced variants occurs during development, as well as under physiological and pathological conditions. 9,12,14,15,47,48 In rat uterus, MYPT1 mRNA levels are decreased during the later stages of pregnancy. 15 Hypoxia exposure increases MYPT1 protein levels in the rat aorta.…”

“…8 The exclusion of the 31-bp 3 0 exon shifts the reading frame and results in a C-terminal leucine zipper (LZ) motif, while exon inclusion encodes a MYPT1 lacking the LZ motif. 9 In vitro and in vivo evidence showed that the LZ motif present in the C-terminal of MYPT1 is required for the activation of MP by type I PKG. [10][11][12] The sensitivity of smooth muscle to cGMP/PKG-mediated relaxation is proportional to the expression ratio of LZ þ / LZ À MYPT1.…”