PKB-Mediated Thr649 Phosphorylation of AS160/TBC1D4 Regulates the R-Wave Amplitude in the Heart

Abstract

The Rab GTPase activating protein (RabGAP), AS160/TBC1D4, is an important substrate of protein kinase B (PKB), and regulates insulin-stimulated trafficking of glucose transporter 4. Besides, AS160/TBC1D4 has also been shown to regulate trafficking of many other membrane proteins including FA translocase/CD36 in cardiomyocytes. However, it is not clear whether it plays any role in regulating heart functions in vivo. Here, we found that PKB-mediated phosphorylation of Thr649 on AS160/TBC1D4 represented one of the major PAS-binding signals in the heart in response to insulin. Mutation of Thr649 to a non-phosphorylatable alanine increased the R-wave amplitude in the AS160Thr649Ala knockin mice. However, this knockin mutation did not affect the heart functions under both normal and infarct conditions. Interestingly, myocardial infarction induced the expression of a related RabGAP, TBC1D1, in the infarct zone as well as in the border zone. Together, these data show that the Thr649 phosphorylation of AS160/TBC1D4 plays an important role in the heart's electrical conduction system through regulating the R-wave amplitude.

Fig 1. The expression of AS160/TBC1D4 and GLUT4 in the four chambers of mouse heart.

A, Immunoblotting analyses of AS160/TBC1D4 and GLUT4 in the lysates (40 μg) of the four chambers of mouse heart. GAPDH was used as a loading control. B, Quantitation of AS160/TBC1D4 expression in the four chambers of mouse heart. n = 6. Asterisk indicates p < 0.05 (atrium versus ventricle). ♯ indicates p < 0.05 (right atrium versus left atrium). C, Quantitation of GLUT4 expression in the four chambers of mouse heart. n = 6. Asterisk indicates p < 0.05 (left ventricle versus the other three chambers).

Fig 2. Phosphorylation of AS160/TBC1D4 in mouse heart in response to insulin.

PAS-binding signals, phosphorylation of AS160-Thr⁶⁴⁹ and PKB-Ser⁴⁷³, and total AS160 and PKB were detected in the lysates (40 μg) of hearts from the wild-type and AS160^Thr649Ala knockin mice that were intraperitoneally injected with PBS (basal) or insulin (150 mU per kg of body weight) for 20 min. GAPDH was used as a loading control.

Fig 3. Cardiac functions in the wild-type and AS160^Thr649Ala knockin mice before and after myocardial infarction.

A, ECG was measured before, and 4 weeks after myocardial infarction in the wild-type and AS160^Thr649Ala knockin mice. Representative ECG graphs were shown. B, Cardiac functions including EF and FS were measured via echocardiography before, and 2 and 4 weeks after myocardial infarction in the wild-type and AS160^Thr649Ala knockin mice. Asterisk indicates p < 0.05. C, Masson’s staining of the infarct heart from the wild-type and AS160^Thr649Ala knockin mice 4 weeks after myocardial infarction. D, Quantitation of the infarction areas detected by Masson’s staining. n = 3.

Fig 4. Expression and phosphorylation of AS160 and TBC1D1 in the infarct heart.

Immunoblotting analyses of phosphorylation of AS160-Thr⁶⁴⁹ and PKB-Ser⁴⁷³, and total AS160, TBC1D1, PKB, GLUT4 and cTnT in the lysates (40 μg) of the infarct zone (I), border zone (B) and remote zone (R) of mouse heart 4 weeks after myocardial infarction. Flotillin-1 was used as a loading control. A, representative blots. B, quantitation of TBC1D1 signals, n = 4. Asterisk indicates p < 0.05 (infarct zone versus remote zone).