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

Allison M. Given, Ozgur Ogut, Frank V. Brozovich

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

During nitric oxide signaling, type Iα cGMP-dependent protein kinase (PKGIα) activates myosin light chain (MLC) phosphatase through an interaction with the 130-kDa myosin targeting subunit (MYPT1), leading to dephosphorylation of 20-kDa MLC and vasodilatation. It has been suggested that the MYPT1-PKGIα interaction is mediated by the COOH-terminal leucine zipper (LZ) of MYPT1 and the NH 2-terminal LZ of PKGIα (HK Surks and ME Mendelsohn. Cell Signal 15: 937-944, 2003; HK Surks et al. Science 286: 1583-1587, 1999), but we previously showed that PKGIα interacts with LZ-positive (LZ+) and LZ-negative (LZ-) MYPT1 isoforms (13). Interestingly, PKGIα 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 LZ+ and LZ- isoforms. Thus, to localize the domain of MYPT1 important for the MYPT1-PKGIα 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 PKGIα in avian smooth muscle tissue lysates. Furthermore, mutations of the RK motif at aa 916-917 (R 916K 917) to AA decreased binding of MYPT1 to PKGIα in chicken gizzard lysates; these mutations had no effect on binding in chicken aorta lysates. However, mutation of R 916K 917 to E 916E 917 eliminated binding, suggesting that one factor important for the PKGIα-MYPT1 interaction is the charge at aa 916-917. These results suggest that, during cGMP-mediated signaling, aa 888-928 of MYPT1 mediate the PKGIα-MYPT1 interaction.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume292
Issue number1
DOIs
StatePublished - Jan 2007

Fingerprint

Leucine Zippers
Mutation
Cyclic GMP-Dependent Protein Kinase Type I
Chickens
Protein Isoforms
Myosin-Light-Chain Phosphatase
Avian Gizzard
Myosin Light Chains
Myosins
Vasodilation
Smooth Muscle
Muscle
Aorta
Nitric Oxide

Keywords

  • Calcium desensitization
  • cGMP
  • cGMP-dependent protein kinase
  • Myosin light chain phosphatase
  • Nitric oxide
  • Smooth muscle

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology
  • Physiology

Cite this

MYPT1 mutants demonstrate the importance of aa 888-928 for the interaction with PKGIα. / Given, Allison M.; Ogut, Ozgur; Brozovich, Frank V.

In: American Journal of Physiology - Cell Physiology, Vol. 292, No. 1, 01.2007.

Research output: Contribution to journalArticle

@article{ca7c9e9b9e294013adc2acb8f70313c2,
title = "MYPT1 mutants demonstrate the importance of aa 888-928 for the interaction with PKGIα",
abstract = "During nitric oxide signaling, type Iα cGMP-dependent protein kinase (PKGIα) activates myosin light chain (MLC) phosphatase through an interaction with the 130-kDa myosin targeting subunit (MYPT1), leading to dephosphorylation of 20-kDa MLC and vasodilatation. It has been suggested that the MYPT1-PKGIα interaction is mediated by the COOH-terminal leucine zipper (LZ) of MYPT1 and the NH 2-terminal LZ of PKGIα (HK Surks and ME Mendelsohn. Cell Signal 15: 937-944, 2003; HK Surks et al. Science 286: 1583-1587, 1999), but we previously showed that PKGIα interacts with LZ-positive (LZ+) and LZ-negative (LZ-) MYPT1 isoforms (13). Interestingly, PKGIα 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 LZ+ and LZ- isoforms. Thus, to localize the domain of MYPT1 important for the MYPT1-PKGIα 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 PKGIα in avian smooth muscle tissue lysates. Furthermore, mutations of the RK motif at aa 916-917 (R 916K 917) to AA decreased binding of MYPT1 to PKGIα in chicken gizzard lysates; these mutations had no effect on binding in chicken aorta lysates. However, mutation of R 916K 917 to E 916E 917 eliminated binding, suggesting that one factor important for the PKGIα-MYPT1 interaction is the charge at aa 916-917. These results suggest that, during cGMP-mediated signaling, aa 888-928 of MYPT1 mediate the PKGIα-MYPT1 interaction.",
keywords = "Calcium desensitization, cGMP, cGMP-dependent protein kinase, Myosin light chain phosphatase, Nitric oxide, Smooth muscle",
author = "Given, {Allison M.} and Ozgur Ogut and Brozovich, {Frank V.}",
year = "2007",
month = "1",
doi = "10.1152/ajpcell.00175.2006",
language = "English (US)",
volume = "292",
journal = "American Journal of Physiology - Renal Fluid and Electrolyte Physiology",
issn = "1931-857X",
publisher = "American Physiological Society",
number = "1",

}

TY - JOUR

T1 - MYPT1 mutants demonstrate the importance of aa 888-928 for the interaction with PKGIα

AU - Given, Allison M.

AU - Ogut, Ozgur

AU - Brozovich, Frank V.

PY - 2007/1

Y1 - 2007/1

N2 - During nitric oxide signaling, type Iα cGMP-dependent protein kinase (PKGIα) activates myosin light chain (MLC) phosphatase through an interaction with the 130-kDa myosin targeting subunit (MYPT1), leading to dephosphorylation of 20-kDa MLC and vasodilatation. It has been suggested that the MYPT1-PKGIα interaction is mediated by the COOH-terminal leucine zipper (LZ) of MYPT1 and the NH 2-terminal LZ of PKGIα (HK Surks and ME Mendelsohn. Cell Signal 15: 937-944, 2003; HK Surks et al. Science 286: 1583-1587, 1999), but we previously showed that PKGIα interacts with LZ-positive (LZ+) and LZ-negative (LZ-) MYPT1 isoforms (13). Interestingly, PKGIα 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 LZ+ and LZ- isoforms. Thus, to localize the domain of MYPT1 important for the MYPT1-PKGIα 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 PKGIα in avian smooth muscle tissue lysates. Furthermore, mutations of the RK motif at aa 916-917 (R 916K 917) to AA decreased binding of MYPT1 to PKGIα in chicken gizzard lysates; these mutations had no effect on binding in chicken aorta lysates. However, mutation of R 916K 917 to E 916E 917 eliminated binding, suggesting that one factor important for the PKGIα-MYPT1 interaction is the charge at aa 916-917. These results suggest that, during cGMP-mediated signaling, aa 888-928 of MYPT1 mediate the PKGIα-MYPT1 interaction.

AB - During nitric oxide signaling, type Iα cGMP-dependent protein kinase (PKGIα) activates myosin light chain (MLC) phosphatase through an interaction with the 130-kDa myosin targeting subunit (MYPT1), leading to dephosphorylation of 20-kDa MLC and vasodilatation. It has been suggested that the MYPT1-PKGIα interaction is mediated by the COOH-terminal leucine zipper (LZ) of MYPT1 and the NH 2-terminal LZ of PKGIα (HK Surks and ME Mendelsohn. Cell Signal 15: 937-944, 2003; HK Surks et al. Science 286: 1583-1587, 1999), but we previously showed that PKGIα interacts with LZ-positive (LZ+) and LZ-negative (LZ-) MYPT1 isoforms (13). Interestingly, PKGIα 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 LZ+ and LZ- isoforms. Thus, to localize the domain of MYPT1 important for the MYPT1-PKGIα 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 PKGIα in avian smooth muscle tissue lysates. Furthermore, mutations of the RK motif at aa 916-917 (R 916K 917) to AA decreased binding of MYPT1 to PKGIα in chicken gizzard lysates; these mutations had no effect on binding in chicken aorta lysates. However, mutation of R 916K 917 to E 916E 917 eliminated binding, suggesting that one factor important for the PKGIα-MYPT1 interaction is the charge at aa 916-917. These results suggest that, during cGMP-mediated signaling, aa 888-928 of MYPT1 mediate the PKGIα-MYPT1 interaction.

KW - Calcium desensitization

KW - cGMP

KW - cGMP-dependent protein kinase

KW - Myosin light chain phosphatase

KW - Nitric oxide

KW - Smooth muscle

UR - http://www.scopus.com/inward/record.url?scp=33846323255&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33846323255&partnerID=8YFLogxK

U2 - 10.1152/ajpcell.00175.2006

DO - 10.1152/ajpcell.00175.2006

M3 - Article

C2 - 16870832

AN - SCOPUS:33846323255

VL - 292

JO - American Journal of Physiology - Renal Fluid and Electrolyte Physiology

JF - American Journal of Physiology - Renal Fluid and Electrolyte Physiology

SN - 1931-857X

IS - 1

ER -