Identification of a novel phosphorylation site on TBC1D4 regulated by AMP-activated protein kinase in skeletal muscle

Jonas Thue Treebak; Eric B. Taylor; Carol A. Witczak; Ding An; Taro Toyoda; Ho-Jin Koh; Jianxin Xie; Edward P. Feener; Jørgen Wojtaszewski; Michael F. Hirshman; Laurie J. Goodyear

doi:10.1152/ajpcell.00297.2009

Identification of a novel phosphorylation site on TBC1D4 regulated by AMP-activated protein kinase in skeletal muscle

Jonas Thue Treebak, Eric B. Taylor, Carol A. Witczak, Ding An, Taro Toyoda, Ho-Jin Koh, Jianxin Xie, Edward P. Feener, Jørgen Wojtaszewski, Michael F. Hirshman, Laurie J. Goodyear

72 Citations (Scopus)

Abstract

TBC1D4 (also known as AS160) regulates glucose transporter 4 (GLUT4) translocation and glucose uptake in adipocytes and skeletal muscle. Its mode of action involves phosphorylation of serine (S)/threonine (T) residues by upstream kinases resulting in inactivation of Rab-GTPase-activating protein (Rab-GAP) activity leading to GLUT4 mobilization. The majority of known phosphorylation sites on TBC1D4 lie within the Akt consensus motif and are phosphorylated by insulin stimulation. However, the 5′-AMP-activated protein kinase (AMPK) and other kinases may also phosphorylate TBC1D4, and therefore we hypothesized the presence of additional phosphorylation sites. Mouse skeletal muscles were contracted or stimulated with 5-aminoimidazole-4-carboxamide-1-β-D- ribofuranoside (AICAR), and muscle lysates were subjected to mass spectrometry analyses resulting in identification of novel putative phosphorylation sites on TBC1D4. The surrounding amino acid sequence predicted that S711 would be recognized by AMPK. Using a phosphospecific antibody against S711, we found that AICAR and contraction increased S711 phosphorylation in mouse skeletal muscle, and this increase was abolished in muscle-specific AMPKα2 kinase-dead transgenic mice. Exercise in human vastus lateralis muscle also increased TBC1D4 S711 phosphorylation. Recombinant AMPK, but not Akt1, Akt2, or PKCζ, phosphorylated purified muscle TBC1D4 on S711 in vitro. Interestingly, S711 was also phosphorylated in response to insulin in an Akt2- and rapamycinindependent, but a wortmannin-sensitive, manner, suggesting this site is regulated by one or more additional upstream kinases. Despite increased S711 phosphorylation with AICAR, contraction, and insulin, mutation of S711 to alanine did not alter glucose uptake in response to these stimuli. S711 is a novel TBC1D4 phosphorylation site regulated by AMPK in skeletal muscle.

Original language	English
Journal	American Journal of Physiology: Cell Physiology
Volume	298
Issue number	2
Pages (from-to)	C377-C385
Number of pages	9
ISSN	0363-6143
DOIs	https://doi.org/10.1152/ajpcell.00297.2009
Publication status	Published - Feb 2010

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Treebak, J. T., Taylor, E. B., Witczak, C. A., An, D., Toyoda, T., Koh, H.-J., Xie, J., Feener, E. P., Wojtaszewski, J., Hirshman, M. F., & Goodyear, L. J. (2010). Identification of a novel phosphorylation site on TBC1D4 regulated by AMP-activated protein kinase in skeletal muscle. American Journal of Physiology: Cell Physiology, 298(2), C377-C385. https://doi.org/10.1152/ajpcell.00297.2009

Treebak, JT, Taylor, EB, Witczak, CA, An, D, Toyoda, T, Koh, H-J, Xie, J, Feener, EP, Wojtaszewski, J, Hirshman, MF & Goodyear, LJ 2010, 'Identification of a novel phosphorylation site on TBC1D4 regulated by AMP-activated protein kinase in skeletal muscle', American Journal of Physiology: Cell Physiology, vol. 298, no. 2, pp. C377-C385. https://doi.org/10.1152/ajpcell.00297.2009

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title = "Identification of a novel phosphorylation site on TBC1D4 regulated by AMP-activated protein kinase in skeletal muscle",

abstract = "TBC1D4 (also known as AS160) regulates glucose transporter 4 (GLUT4) translocation and glucose uptake in adipocytes and skeletal muscle. Its mode of action involves phosphorylation of serine (S)/threonine (T) residues by upstream kinases resulting in inactivation of Rab-GTPase-activating protein (Rab-GAP) activity leading to GLUT4 mobilization. The majority of known phosphorylation sites on TBC1D4 lie within the Akt consensus motif and are phosphorylated by insulin stimulation. However, the 5′-AMP-activated protein kinase (AMPK) and other kinases may also phosphorylate TBC1D4, and therefore we hypothesized the presence of additional phosphorylation sites. Mouse skeletal muscles were contracted or stimulated with 5-aminoimidazole-4-carboxamide-1-β-D- ribofuranoside (AICAR), and muscle lysates were subjected to mass spectrometry analyses resulting in identification of novel putative phosphorylation sites on TBC1D4. The surrounding amino acid sequence predicted that S711 would be recognized by AMPK. Using a phosphospecific antibody against S711, we found that AICAR and contraction increased S711 phosphorylation in mouse skeletal muscle, and this increase was abolished in muscle-specific AMPKα2 kinase-dead transgenic mice. Exercise in human vastus lateralis muscle also increased TBC1D4 S711 phosphorylation. Recombinant AMPK, but not Akt1, Akt2, or PKCζ, phosphorylated purified muscle TBC1D4 on S711 in vitro. Interestingly, S711 was also phosphorylated in response to insulin in an Akt2- and rapamycinindependent, but a wortmannin-sensitive, manner, suggesting this site is regulated by one or more additional upstream kinases. Despite increased S711 phosphorylation with AICAR, contraction, and insulin, mutation of S711 to alanine did not alter glucose uptake in response to these stimuli. S711 is a novel TBC1D4 phosphorylation site regulated by AMPK in skeletal muscle.",

author = "Treebak, {Jonas Thue} and Taylor, {Eric B.} and Witczak, {Carol A.} and Ding An and Taro Toyoda and Ho-Jin Koh and Jianxin Xie and Feener, {Edward P.} and J{\o}rgen Wojtaszewski and Hirshman, {Michael F.} and Goodyear, {Laurie J.}",

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T1 - Identification of a novel phosphorylation site on TBC1D4 regulated by AMP-activated protein kinase in skeletal muscle

AU - Treebak, Jonas Thue

AU - Taylor, Eric B.

AU - Witczak, Carol A.

AU - An, Ding

AU - Toyoda, Taro

AU - Koh, Ho-Jin

AU - Xie, Jianxin

AU - Feener, Edward P.

AU - Wojtaszewski, Jørgen

AU - Hirshman, Michael F.

AU - Goodyear, Laurie J.

N1 - CURIS 2010 5200 013

PY - 2010/2

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N2 - TBC1D4 (also known as AS160) regulates glucose transporter 4 (GLUT4) translocation and glucose uptake in adipocytes and skeletal muscle. Its mode of action involves phosphorylation of serine (S)/threonine (T) residues by upstream kinases resulting in inactivation of Rab-GTPase-activating protein (Rab-GAP) activity leading to GLUT4 mobilization. The majority of known phosphorylation sites on TBC1D4 lie within the Akt consensus motif and are phosphorylated by insulin stimulation. However, the 5′-AMP-activated protein kinase (AMPK) and other kinases may also phosphorylate TBC1D4, and therefore we hypothesized the presence of additional phosphorylation sites. Mouse skeletal muscles were contracted or stimulated with 5-aminoimidazole-4-carboxamide-1-β-D- ribofuranoside (AICAR), and muscle lysates were subjected to mass spectrometry analyses resulting in identification of novel putative phosphorylation sites on TBC1D4. The surrounding amino acid sequence predicted that S711 would be recognized by AMPK. Using a phosphospecific antibody against S711, we found that AICAR and contraction increased S711 phosphorylation in mouse skeletal muscle, and this increase was abolished in muscle-specific AMPKα2 kinase-dead transgenic mice. Exercise in human vastus lateralis muscle also increased TBC1D4 S711 phosphorylation. Recombinant AMPK, but not Akt1, Akt2, or PKCζ, phosphorylated purified muscle TBC1D4 on S711 in vitro. Interestingly, S711 was also phosphorylated in response to insulin in an Akt2- and rapamycinindependent, but a wortmannin-sensitive, manner, suggesting this site is regulated by one or more additional upstream kinases. Despite increased S711 phosphorylation with AICAR, contraction, and insulin, mutation of S711 to alanine did not alter glucose uptake in response to these stimuli. S711 is a novel TBC1D4 phosphorylation site regulated by AMPK in skeletal muscle.

AB - TBC1D4 (also known as AS160) regulates glucose transporter 4 (GLUT4) translocation and glucose uptake in adipocytes and skeletal muscle. Its mode of action involves phosphorylation of serine (S)/threonine (T) residues by upstream kinases resulting in inactivation of Rab-GTPase-activating protein (Rab-GAP) activity leading to GLUT4 mobilization. The majority of known phosphorylation sites on TBC1D4 lie within the Akt consensus motif and are phosphorylated by insulin stimulation. However, the 5′-AMP-activated protein kinase (AMPK) and other kinases may also phosphorylate TBC1D4, and therefore we hypothesized the presence of additional phosphorylation sites. Mouse skeletal muscles were contracted or stimulated with 5-aminoimidazole-4-carboxamide-1-β-D- ribofuranoside (AICAR), and muscle lysates were subjected to mass spectrometry analyses resulting in identification of novel putative phosphorylation sites on TBC1D4. The surrounding amino acid sequence predicted that S711 would be recognized by AMPK. Using a phosphospecific antibody against S711, we found that AICAR and contraction increased S711 phosphorylation in mouse skeletal muscle, and this increase was abolished in muscle-specific AMPKα2 kinase-dead transgenic mice. Exercise in human vastus lateralis muscle also increased TBC1D4 S711 phosphorylation. Recombinant AMPK, but not Akt1, Akt2, or PKCζ, phosphorylated purified muscle TBC1D4 on S711 in vitro. Interestingly, S711 was also phosphorylated in response to insulin in an Akt2- and rapamycinindependent, but a wortmannin-sensitive, manner, suggesting this site is regulated by one or more additional upstream kinases. Despite increased S711 phosphorylation with AICAR, contraction, and insulin, mutation of S711 to alanine did not alter glucose uptake in response to these stimuli. S711 is a novel TBC1D4 phosphorylation site regulated by AMPK in skeletal muscle.

U2 - 10.1152/ajpcell.00297.2009

DO - 10.1152/ajpcell.00297.2009

M3 - Journal article

C2 - 19923418

SN - 0363-6143

VL - 298

SP - C377-C385

JO - American Journal of Physiology: Cell Physiology

JF - American Journal of Physiology: Cell Physiology

IS - 2

ER -

Identification of a novel phosphorylation site on TBC1D4 regulated by AMP-activated protein kinase in skeletal muscle

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