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

Area of Science:

• * Molecular biology
• * Cellular metabolism
• * Exercise physiology

Background:

• * TBC1D4 (AS160) is a key regulator of glucose transporter 4 (GLUT4) translocation and glucose uptake in muscle and fat cells.
• * Its activity is modulated by phosphorylation, primarily at Akt consensus sites upon insulin stimulation.
• * The role of other kinases, like AMPK, in TBC1D4 phosphorylation and its impact on glucose metabolism remains incompletely understood.

Purpose of the Study:

• * To investigate novel phosphorylation sites on TBC1D4 beyond known Akt-mediated sites.
• * To determine the role of AMPK in phosphorylating TBC1D4, particularly in response to muscle contraction and metabolic stimuli.
• * To elucidate the functional consequence of TBC1D4 S711 phosphorylation on glucose uptake.

Main Methods:

• * Mass spectrometry was used to identify novel phosphorylation sites on TBC1D4 in mouse skeletal muscle stimulated by contraction or AICAR.
• * Phosphospecific antibodies targeting S711 were employed to assess phosphorylation changes in response to stimuli and in AMPK knockout models.
• * In vitro kinase assays using recombinant AMPK and Akt isoforms were performed.
• * Site-directed mutagenesis (S711A) was used to evaluate the functional impact on glucose uptake.

Main Results:

• * Novel putative phosphorylation sites on TBC1D4 were identified, with S711 predicted to be an AMPK target.
• * AICAR stimulation and muscle contraction significantly increased TBC1D4 S711 phosphorylation in mouse and human skeletal muscle.
• * This increase was dependent on AMPK activity, as shown in AMPKalpha2 kinase-dead mice.
• * Recombinant AMPK, but not Akt or PKCzeta, phosphorylated TBC1D4 S711 in vitro.
• * Insulin also induced S711 phosphorylation in an Akt2- and rapamycin-independent, wortmannin-sensitive manner.
• * Despite increased S711 phosphorylation under various stimuli, mutating S711 to alanine did not affect glucose uptake.

Conclusions:

• * S711 represents a novel phosphorylation site on TBC1D4 regulated by AMPK in skeletal muscle.
• * AMPK activation, through exercise or AICAR, leads to TBC1D4 S711 phosphorylation.
• * While S711 is phosphorylated by AMPK and other kinases, its phosphorylation status does not appear to be essential for mediating glucose uptake in response to these stimuli.