The specific phosphorylation site of YAP is of critical importance in determining its activity as a transcriptional co-activator, but is becoming recognized as increasingly complex. Earlier publications focused largely on the well characterized Hippo-mediated YAP phosphorylation at serine 127, resulting in a “Hippo-on” state, which results in cytosolic retention via binding with 14-3-3 proteins, reduced transcriptional activity due to exclusion from the nucleus, and proteasomal degradation.
62 Conversely, “Hippo-off” activates YAP via loss of phosphorylation at s127, disruption of its “stable” cytoplasmic retention, and translocation to the nucleus. Although Hippo-signaling is important in the functional fate of YAP, as a determinant of subcellular location, it is not exclusive, and rather SRC-mediated YAP phosphorylation at tyrosine 357 has been shown to independently promote nuclear translocation regardless of serine 127 phosphorylation status, and further that the same SRC pathway negatively regulates the Hippo pathway to a state of “Hippo-off.”
63 In contrast to previously held theories, several more recent live imaging studies have established that there exists YAP, which is nuclear, yet phosphorylated at serine 127, and that it exists in a state of dynamic fluctuations, revoking prior principles of an oversimplified binary on/off status.
64–66 Recent findings suggest that nuclear export from within the nucleus is the greatest determinant of the subcellular location of YAP, rather than reduced nuclear import secondary to cytoplasmic retention via YAP-14-3-3 binding.
64 Not only is YAP held in the nucleus for longer, (avoiding degradation), but its actual duration of attachment to chromatin is increased when it is phosphorylated at tyrosine 357. Consistent with this concept was our finding that pYAP(y357) was markedly increased on stiffened substrates, total-YAP was predominantly nuclear on stiffened substrates as seen through immunofluorescence, and its transcriptional activity was validated by increased CTGF and cellular proliferation (
Figs. 2 and
3). Equally, lower levels of pYAP(s127) relative to total YAP were observed on stiff substrates, and this corresponded to the nuclear translocation witnessed on immunofluorescence and increased transcriptional output (
Fig. 2E). We found greater differential expression of pYAP(s127) (via Hippo pathway) relative to total YAP, across soft and stiff substrates, when compared to pYAP(y357), perhaps suggesting that the Hippo pathway is implicated more than the SRC-mediated phosphorylation at tyrosine y357 in the mechanosensitive regulation of YAP. We have illustrated these findings in
Figure 4, whereby a “seesaw” balance involving both YAP phosphorylation at serine 127 and tyrosine 357, determines subcellular location, and that there exists constant shuttling in and out of the nucleus, determined by the balance of phosphorylation site (
Fig. 4). A further determinant of YAP activity is through a direct mechanical connection between focal adhesions at the cell membrane and the nucleus, mediated through F-actin cytoskeleton.
66 Exposure to stiffened microenvironment is transmitted to the nucleus, resulting in nuclear flattening, stretching of the nuclear pores, reducing their resistance to molecular transport and increasing YAP nuclear import.
66 This is consistent with our immunofluorescence findings of NLC cell culture on 4 kPA versus 100 kPA substrates with increased F-actin and nuclear YAP being visible across all cells on stiffened substrates (
Fig. 3).