2. Part 2: MG53 ligase ubiquitinates focal adhesion kinase during skeletal
2.1. FAK protein is down-regulated during skeletal myogenesis
I first found that there are conflicting data regarding the expression level of FAK during skeletal myogenesis. For example, the FAK expression level gradually decreases during myogenesis in primary mouse myoblast cultures but remains constant during C2C12 myogenesis (Figures I and II) (23-25).
Figure I. Expression of FAK during myogenic differentiation in primary myoblast cultures. Adopted from (24) Focal Adhesion Kinase Signaling Regulates
the Expression of Caveolin 3 and beta 1 Integrin, Genes Essential for Normal
(A) Mouse primary myoblasts were cultured in differentiation medium and observed by phase contrast microscopy. Multinucleated myotubes formed within 48 h of initiation of differentiation. Myotube maturation is associated with the appearance of aligned sarcomeric striations and myotube contractions (seen at high magnification at 96 h). Bar, 100 μm (except for 96 h, high-magnification panel, 20 μm).
(B) Primary myoblasts were cultured in differentiation medium then analyzed by immunoblots. The levels of FAK and phosphorylated FAK were high during early differentiation then decreased as myotubes mature. Myogenin was transiently up-regulated during early differentiation, whereas sarcomeric α-actin increased dramatically with myogenic differentiation.
Adopted from (24) Focal Adhesion Kinase Signaling Regulates the Expression of Caveolin 3 and beta 1 Integrin, Genes Essential for Normal Myoblast Fusion. Molecular Biology of the Cell 20 (2009), 3422-3435.
Figure II. FAK phosphorylation at Tyr-397 during myogenic differentiation.
Adopted from (25) Differentiation of C2C12 myoblasts is critically regulated by FAK signaling.
American Journal of Physiology – Regulatory, Integrative and Comparative Physiology 289 (2005), R862-870.
(A) Representative blots and densitometric readings showing the average values (5 experiments) of percent changes in the amount of FAK, detected with anti-pFAK antibody, in C2C12 cells in serum-starved medium (up to 5 days) compared with proliferating cells.
(B) Control experiments performed with withdrawal and replacement of proliferating medium. Immunoblots (IB) are representative of 3 different experiments. C, extracts from cells cultured in proliferating medium; W, extracts
from washed cells obtained 2 h after the washing procedure. *P < 0.05 compared with proliferating cells (C).
Adopted from Adopted from (25) Differentiation of C2C12 myoblasts is critically regulated by FAK signaling. American Journal of Physiology – Regulatory, Integrative and Comparative Physiology 289 (2005), R862-870.
To reconcile this difference, I re-evaluated the level of FAK expression during C2C12 myogenesis. As characterized before, C2C12 murine myoblasts, used as a model of skeletal muscle development, fuse into multinucleated myotubes when shifted from the growth medium to differentiation medium (26, 27). To promote differentiation, about 90-100% confluent C2C12 cells were kept in a low serum medium for up to 9 days.
Immunoblot analysis of C2C12 protein extracts of each day during differentiation revealed a significant decline in FAK protein expression during C2C12 myogenesis (Figure 1a). After day 3, the FAK protein level gradually decreased and reached a low level after 5 days. The detection of FAK was concomitant with that of proteins proved to be myogenic markers including myosin heavy chain (MyHC), myogenin (Mgn), caveolin 3 (Cav-3) and MyoD (28-32).
and caveolin-3 (Cav-3) were gradually increased during myogenesis, indicating that skeletal myogenesis was well induced.
Murine C2C12 muscle cells
0 1 2 3 4 5 6 7 8 9
MyHC Mgn Cav-3 UBE2H FAK pFAK MG53
GAPDH
a Dif f erentiation days
MyHC FAK 0 1 2 3 4 5 6 7 8 9 10
GAPDH
b Dif f erentiation days
Figure 1. The protein expression level of FAK decreases
Mouse embryonic fibroblasts (MEFs)
p-FAK FAK
Cav-3 Mgn MG53
MyoD MyHC
GAPDH UBE2H 0 1 3 5 7
c Dif f erentiation days
0 1 3 5 7
d Dif f erentiation days
MyHC FAK
GAPDH
Figure 1. The protein expression level of FAK decreases
(a and b) C2C12 myoblasts were differentiated into myotubes for the indicated days. The expression levels of FAK, phospho-FAK (p-FAK) at Tyr- 576/577, MG53, UBE2H, MyHC, Mgn, and Cav-3 were determined by immunoblotting using GAPDH as a loading control (a).
The mRNA levels of FAK and MyHC were determined by RT-PCR using GAPDH as a loading control (b).
(c and d) MEFs were infected with adenoviral MyoD for 12 h and differentiated into myotubes for the indicated number of days. The protein levels of FAK, p-FAK, MG53, UBE2H, MyHC, Mgn, Cav-3, and GAPDH were determined by immunoblotting (c), and the mRNA levels of FAK, MyHC, and GAPDH were determined by RT-PCR (d).
Interestingly, the E2 enzyme UBE2H and the E3 enzyme MG53 were up- regulated during myogenesis. Although the FAK protein level was down-regulated, its mRNA level was nearly constant throughout C2C12 myogenesis (Figure 1b).
We also reconfirmed the decrease in the FAK expression level during MyoD- driven myogenesis in mouse embryonic fibroblasts (MEFs). After adenoviral overexpression of MyoD, the MEFs were induced to differentiate into myotubes.
As shown in Figure 1c, the levels of myogenic marker proteins, such as MyHC,
overexpression led to myogenesis induction. On days 5 and 7, the FAK expression level was decreased greatly, whereas its mRNA level was constant in the MyoD- induced myotubes of MEFs. In addition, UBE2H and MG53 were up-regulated during MyoD-induced myogenesis of MEFs. The data in Figures 1a-d indicate that FAK is degraded during skeletal myogenesis in C2C12 cells and MyoD- overexpressing MEFs.