Characterization of Phosphopeptide Positional Isomers on the Transcriptional Co-activator TAZ

The transcriptional co-activator
with the PDZ binding motif (TAZ)
is a critical regulator of numerous cellular processes such as cell
differentiation, development, proliferation, and cell growth. Aberrant
expression and activity of TAZ are also featured in many human malignancies.
A hallmark of TAZ biology is its cytoplasmic retention mediated by
14-3-3 isoforms in response to phosphorylation of Ser89 by members of the LATS family of kinases. Following the observation
that TAZ is a highly phosphorylated protein even when Ser89 is mutated, high-resolution mass spectrometry employing data-independent
acquisition and ion mobility separation was conducted to elucidate
additional TAZ phosphorylation sites that may play a role in regulating
this critical transcriptional rheostat. Numerous phosphorylation sites
on TAZ were identified, including several novel modifications. Of
notable interest was the identification of positional phosphoisomers
on a phosphopeptide containing Ser89. Optimized use of
a so-called wideband enhancement acquisition technique yielded higher-quality
fragmentation data that confirmed the detection of Ser93 as the positional phosphoisomer partner of Ser89 and
identified diagnostic fragment ions for the phosphorylation events.
Functional analysis indicated that Ser93 phosphorylation
reduces the level of 14-3-3 association and increases the level of
nuclear translocation, indicating this phosphorylation event attenuates
the 14-3-3-mediated TAZ cytoplasmic retention mechanism. These findings
suggest that the biological activities of TAZ are likely dynamically
regulated by multisite phosphorylation.