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.