MYPT1 mutants demonstrate the importance of aa 888–928 for the interaction with PKGIα

Abstract

286: 1583-1587, 1999), but we previously showed that PKGI␣ interacts with LZ-positive (LZϩ) and LZ-negative (LZϪ) MYPT1 isoforms (13). Interestingly, PKGI␣ is known to preferentially bind to RR and RK motifs (WR Dostmann et al. Proc Natl Acad Sci USA 97: 14772-14777, 2000), and there is an RK motif within the aa 888 -928 sequence of MYPT1 in LZϩ and LZϪ isoforms. Thus, to localize the domain of MYPT1 important for the MYPT1-PKGI␣ interaction, we designed four MYPT1 fragments that contained both the aa 888 -928 sequence and the downstream LZ domain (MYPT1FL), lacked both the aa 888 -928 sequence and the LZ domain (MYPT1TR), lacked only the aa 888 -928 sequence (MYPT1SO), or lacked only the LZ domain (MYPT1TR2). Using coimmunoprecipitation, we found that only the fragments containing the aa 888 -928 sequence (MYPT1FL and MYPT1TR2) were able to form a complex with PKGI␣ in avian smooth muscle tissue lysates. Furthermore, mutations of the RK motif at aa 916 -917 (R 916 K 917 ) to AA decreased binding of MYPT1 to PKGI␣ in chicken gizzard lysates; these mutations had no effect on binding in chicken aorta lysates. However, mutation of R -calmodulin activation of MLC kinase has been proposed to be the primary event for regulation of MLC 20 phosphorylation, and MLC phosphatase was thought to be constitutively active (12,22). However, recent studies have shown that MLC phosphatase is an important target for the physiological regulation of vascular tone; MLC phosphatase activity can be stimulated to produce Ca 2ϩ desensitization and inhibited to produce Ca 2ϩ sensitization (reviewed in Refs. 12 and 22).MLC phosphatase is a trimeric enzyme consisting of a 130-kDa myosin targeting subunit (MYPT1), a 37-kDa catalytic subunit (PP1c␦), and a 20-kDa subunit of unknown function (M20) (12). MYPT1, the primary site for regulation of phosphatase activity, has multiple binding and phosphorylation sites, which allow its function to be tightly controlled. Multiple pathways feed into this regulation, including G protein-coupled signaling and nitric oxide (NO) (reviewed in Refs. 12 and 22).NO-mediated vasodilatation is a fundamental response of the vasculature and is the classical model for Ca 2ϩ desensitization (9, 10). NO diffuses into the smooth muscle cells and activates guanylate cyclase, thereby increasing the production of cGMP. This second messenger can then bind to and activate type I␣ cGMP-dependent protein kinase (PKGI␣), which interacts with multiple targets within the smooth muscle cell, including the L-type Ca 2ϩ channel (8), maxi-K ϩ channels (1), sarcoplasmic reticulum channels (20), telokin (4, 25), and MYPT1 (13,23,24), all of which have been demonstrated to produce smooth muscle relaxation.MYPT1 (Fig. 1A) contains two putative COOH-terminal coiled-coil (CC) domains at aa 715-746 and aa 929 -969, which have 60% and 81% probabilities of CC formation, respectively (16), and a third domain at aa 1013-1039, which is known as the leucine zipper (LZ) (12). These sequences are numbered according to the M133 MYPT1 is...

Phosphorylation-dependent Autoinhibition of Myosin Light Chain Phosphatase Accounts for Ca2+ Sensitization Force of Smooth Muscle Contraction

“…In addition, an unidentified Thr-696 phosphatase is possibly activated in response to the cGMP signal. PKG is reported to associate with the C-terminal domain of MYPT1 and is involved in SM relaxation (23,25). The phosphorylation of telokin by PKG activates MLCP through an unknown mechanism (42,65).…”

“…An exon 13 splicing of MYPT1 is involved in Ca 2ϩ sensitization that occurs in response to GTP (21), whereas a splice variant of MYPT1, containing the C-terminal Leu-zipper sequence, correlates with cGMP-dependent relaxation of smooth muscle (22). Direct binding of PKG to MYPT1 at the Leu-zipper domain and/or Arg/Lys-rich domain is involved in the activation of MLCP (23)(24)(25). In addition, a myosin phosphatase-Rho interacting protein (M-RIP) is directly associated with the MYPT1 C-terminal domain, proposed to recruit RhoA to the MLCP complex (26).…”

Interactions between the Leucine-zipper Motif of cGMP-Dependent Protein Kinase and the C-terminal Region of the Targeting Subunit of Myosin Light Chain Phosphatase

“…33 For their studies, Given et al33 produced four constructs: MYPT1FL, MYPT1TR, MYPT1SO and MYPT1TR2 (the sequences of these constructs and their equivalent designations according to our scheme are shown in Table 2). Our finding that E 2 CCLZ binds N-PKG (row 1, Table 2) and that of Surks et al that AL9 binds to PKG (row 10) are compatible with the finding of Given et al that MYPT1FL co-immunoprecipitates with PKG (row 16).…”

IRAG and novel PKG targeting in the cardiovascular system

“…Two diverse MYPT1 isoforms are expressed in vascular smooth muscles, depending on the developmental stage: a leucine zipper isoform (LZ ϩ ) containing a COOH-terminal leucine zipper motif that binds to PKG1␣ and sensitizes cGMP-dependent relaxation, a leucine zipper-deficient isoform that is cGMP insensitive (63). The LZ-deficient form of MYPT1 binds via its N1-N2 coiled-coil domain to PKG1␣, forming a heterotetramer (29,47) (Fig. 3E).…”