Phosphorylation and Mutation of Human Cardiac Troponin I Deferentially Destabilize the Interaction of the Functional Regions of Troponin I with Troponin C

Monica X. Li, Xu Wang, Darrin A. Lindhout, Nina Buscemi, Jennifer E. Van Eyk, Brian D. Sykes

Research output: Contribution to journalArticle

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Abstract

We have utilized 2D {1H,15N}-HSQC NMR spectroscopy to elucidate the binding of three segments of cTnI in native, phosphorylated, and mutated states to cTnC. The near N-terminal region (cRp; residues 34-71) contains the protein kinase C (PKC) phosphorylation sites S41 and S43, the inhibitory region (cIp; residues 128-147) contains another PKC site T142 and a familial hypertrophic cardiomyopathy (FHC) mutation R144G, and the switch region (cSp; residues 147-163) contains the novel p21-activated kinase (PAK) site S149 and another FHC mutation R161W. While S41/S43 phosphorylation of cRp had minimal disruption in the interaction of cRp and cTnC·3Ca 2+, T142 phosphorylation reduced the affinity of cIp for cCTnC·2Ca2+ by ∼14-fold and S149 phosphorylation reduced the affinity of cSp for cNTnC·Ca2+ by ∼10-fold. The mutation R144G caused a ∼6-fold affinity decrease of cIp for cCTnC·2Ca2+ and mutation R161W destabilized the interaction of cSp and cNTnC· Ca2+ by ∼1.4-fold. When cIp was both T142 phosphorylated and R144G mutated, its affinity for cCTnC· 2Ca 2+ was reduced ∼19-fold, and when cSp was both S149 phosphorylated and R161W mutated, its affinity for cNTnC·Ca2+ was reduced ∼4-fold. Thus, while the FHC mutation R144G enhances the effect of T142 phosphorylation on the interaction of cIp and cCTnC·2Ca 2+, the FHC mutation R161W suppresses the effect of S149 phosphorylation on the interaction of cSp and cNTnC·Ca2-, demonstrating linkages between the FHC mutation and phosphorylation of cTnI. The observed alterations corroborate well with structural data. These results suggest that while the modifications in the cRp region have minimal influence, those in the key functional cIp-cSp region have a pronounced effect on the interaction of cTnI and cTnC, which may correlate with the altered myofilament function and cardiac muscle contraction under pathophysiological conditions.

Original languageEnglish (US)
Pages (from-to)14460-14468
Number of pages9
JournalBiochemistry
Volume42
Issue number49
DOIs
StatePublished - Dec 16 2003
Externally publishedYes

Fingerprint

Troponin C
Phosphorylation
Troponin I
Familial Hypertrophic Cardiomyopathy
Mutation
sprodiamide
Protein Kinase C
p21-Activated Kinases
Myofibrils
Muscle Contraction
Nuclear magnetic resonance spectroscopy
Muscle
Myocardium
Magnetic Resonance Spectroscopy
Switches

ASJC Scopus subject areas

  • Biochemistry

Cite this

Phosphorylation and Mutation of Human Cardiac Troponin I Deferentially Destabilize the Interaction of the Functional Regions of Troponin I with Troponin C. / Li, Monica X.; Wang, Xu; Lindhout, Darrin A.; Buscemi, Nina; Van Eyk, Jennifer E.; Sykes, Brian D.

In: Biochemistry, Vol. 42, No. 49, 16.12.2003, p. 14460-14468.

Research output: Contribution to journalArticle

Li, Monica X. ; Wang, Xu ; Lindhout, Darrin A. ; Buscemi, Nina ; Van Eyk, Jennifer E. ; Sykes, Brian D. / Phosphorylation and Mutation of Human Cardiac Troponin I Deferentially Destabilize the Interaction of the Functional Regions of Troponin I with Troponin C. In: Biochemistry. 2003 ; Vol. 42, No. 49. pp. 14460-14468.
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abstract = "We have utilized 2D {1H,15N}-HSQC NMR spectroscopy to elucidate the binding of three segments of cTnI in native, phosphorylated, and mutated states to cTnC. The near N-terminal region (cRp; residues 34-71) contains the protein kinase C (PKC) phosphorylation sites S41 and S43, the inhibitory region (cIp; residues 128-147) contains another PKC site T142 and a familial hypertrophic cardiomyopathy (FHC) mutation R144G, and the switch region (cSp; residues 147-163) contains the novel p21-activated kinase (PAK) site S149 and another FHC mutation R161W. While S41/S43 phosphorylation of cRp had minimal disruption in the interaction of cRp and cTnC·3Ca 2+, T142 phosphorylation reduced the affinity of cIp for cCTnC·2Ca2+ by ∼14-fold and S149 phosphorylation reduced the affinity of cSp for cNTnC·Ca2+ by ∼10-fold. The mutation R144G caused a ∼6-fold affinity decrease of cIp for cCTnC·2Ca2+ and mutation R161W destabilized the interaction of cSp and cNTnC· Ca2+ by ∼1.4-fold. When cIp was both T142 phosphorylated and R144G mutated, its affinity for cCTnC· 2Ca 2+ was reduced ∼19-fold, and when cSp was both S149 phosphorylated and R161W mutated, its affinity for cNTnC·Ca2+ was reduced ∼4-fold. Thus, while the FHC mutation R144G enhances the effect of T142 phosphorylation on the interaction of cIp and cCTnC·2Ca 2+, the FHC mutation R161W suppresses the effect of S149 phosphorylation on the interaction of cSp and cNTnC·Ca2-, demonstrating linkages between the FHC mutation and phosphorylation of cTnI. The observed alterations corroborate well with structural data. These results suggest that while the modifications in the cRp region have minimal influence, those in the key functional cIp-cSp region have a pronounced effect on the interaction of cTnI and cTnC, which may correlate with the altered myofilament function and cardiac muscle contraction under pathophysiological conditions.",
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T1 - Phosphorylation and Mutation of Human Cardiac Troponin I Deferentially Destabilize the Interaction of the Functional Regions of Troponin I with Troponin C

AU - Li, Monica X.

AU - Wang, Xu

AU - Lindhout, Darrin A.

AU - Buscemi, Nina

AU - Van Eyk, Jennifer E.

AU - Sykes, Brian D.

PY - 2003/12/16

Y1 - 2003/12/16

N2 - We have utilized 2D {1H,15N}-HSQC NMR spectroscopy to elucidate the binding of three segments of cTnI in native, phosphorylated, and mutated states to cTnC. The near N-terminal region (cRp; residues 34-71) contains the protein kinase C (PKC) phosphorylation sites S41 and S43, the inhibitory region (cIp; residues 128-147) contains another PKC site T142 and a familial hypertrophic cardiomyopathy (FHC) mutation R144G, and the switch region (cSp; residues 147-163) contains the novel p21-activated kinase (PAK) site S149 and another FHC mutation R161W. While S41/S43 phosphorylation of cRp had minimal disruption in the interaction of cRp and cTnC·3Ca 2+, T142 phosphorylation reduced the affinity of cIp for cCTnC·2Ca2+ by ∼14-fold and S149 phosphorylation reduced the affinity of cSp for cNTnC·Ca2+ by ∼10-fold. The mutation R144G caused a ∼6-fold affinity decrease of cIp for cCTnC·2Ca2+ and mutation R161W destabilized the interaction of cSp and cNTnC· Ca2+ by ∼1.4-fold. When cIp was both T142 phosphorylated and R144G mutated, its affinity for cCTnC· 2Ca 2+ was reduced ∼19-fold, and when cSp was both S149 phosphorylated and R161W mutated, its affinity for cNTnC·Ca2+ was reduced ∼4-fold. Thus, while the FHC mutation R144G enhances the effect of T142 phosphorylation on the interaction of cIp and cCTnC·2Ca 2+, the FHC mutation R161W suppresses the effect of S149 phosphorylation on the interaction of cSp and cNTnC·Ca2-, demonstrating linkages between the FHC mutation and phosphorylation of cTnI. The observed alterations corroborate well with structural data. These results suggest that while the modifications in the cRp region have minimal influence, those in the key functional cIp-cSp region have a pronounced effect on the interaction of cTnI and cTnC, which may correlate with the altered myofilament function and cardiac muscle contraction under pathophysiological conditions.

AB - We have utilized 2D {1H,15N}-HSQC NMR spectroscopy to elucidate the binding of three segments of cTnI in native, phosphorylated, and mutated states to cTnC. The near N-terminal region (cRp; residues 34-71) contains the protein kinase C (PKC) phosphorylation sites S41 and S43, the inhibitory region (cIp; residues 128-147) contains another PKC site T142 and a familial hypertrophic cardiomyopathy (FHC) mutation R144G, and the switch region (cSp; residues 147-163) contains the novel p21-activated kinase (PAK) site S149 and another FHC mutation R161W. While S41/S43 phosphorylation of cRp had minimal disruption in the interaction of cRp and cTnC·3Ca 2+, T142 phosphorylation reduced the affinity of cIp for cCTnC·2Ca2+ by ∼14-fold and S149 phosphorylation reduced the affinity of cSp for cNTnC·Ca2+ by ∼10-fold. The mutation R144G caused a ∼6-fold affinity decrease of cIp for cCTnC·2Ca2+ and mutation R161W destabilized the interaction of cSp and cNTnC· Ca2+ by ∼1.4-fold. When cIp was both T142 phosphorylated and R144G mutated, its affinity for cCTnC· 2Ca 2+ was reduced ∼19-fold, and when cSp was both S149 phosphorylated and R161W mutated, its affinity for cNTnC·Ca2+ was reduced ∼4-fold. Thus, while the FHC mutation R144G enhances the effect of T142 phosphorylation on the interaction of cIp and cCTnC·2Ca 2+, the FHC mutation R161W suppresses the effect of S149 phosphorylation on the interaction of cSp and cNTnC·Ca2-, demonstrating linkages between the FHC mutation and phosphorylation of cTnI. The observed alterations corroborate well with structural data. These results suggest that while the modifications in the cRp region have minimal influence, those in the key functional cIp-cSp region have a pronounced effect on the interaction of cTnI and cTnC, which may correlate with the altered myofilament function and cardiac muscle contraction under pathophysiological conditions.

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