Protein serine/threonine phosphatase 2A (PP2A) is an abundant cellular enzyme with numerous substrates that modulate a wide variety of cellular functions. Considering the multitude of cellular processes under the control of PP2A, it is not surprising that several different mechanisms exist to regulate phosphatase activity. These regulatory mechanisms include association with specific regulatory subunits and post-translational modifications of PP2Ac (i.e. phosphorylation, carboxymethylation, and ubiquitination) (1
). Both biochemical and structural studies of PP2A have provided key mechanistic insights to explicate regulation of phosphatase holoenzyme composition and activity via phosphorylation and carboxymethylation (1
); however, little is known about PP2Ac ubiquitination beyond the initial report demonstrating the polyubiquitination and degradation of microtubule-associated PP2Ac (3
). The E3 ubiquitin ligase responsible for targeting PP2Ac for proteasome degradation is Mid1, a protein linked to the congenital disorder Opitz Syndrome (OS). Human fibroblasts derived from a fetus with OS exhibit a loss of PP2Ac ubiquitination, increased levels of microtubule-associated PP2Ac, and a concomitant decrease in the phosphorylation of general microtubule-associated proteins as compared to age-matched control fibroblasts (3
). Thus it appears that deregulation of PP2Ac ubiquitination culminates in the pathogenesis of OS.
Another key player in Mid1-dependent PP2Ac ubiquitination is alpha4, a mostly alpha-helical protein purported to serve as a scaffold for Mid1 and PP2Ac (3
). Alpha4 contains independent binding sites for PP2Ac and Mid1 on its N- and C-terminus, respectively (3
); yet, its biochemical associations with Mid1 and PP2Ac have been primarily studied independently of one another. Nevertheless, colocalization of alpha4 and exogenous Mid1 at microtubule structures suggests that alpha4 plays an important role in PP2Ac ubiquitination (3
). Another report suggested that alpha4 facilitates dephosphorylation of Mid1 by PP2Ac, as increased alpha4 expression caused a reduction in the cellular levels of phosphorylated Mid1 protein (8
). Indeed, it is reasonable to posit that alpha4 can support cross-regulation of both Mid1 and PP2Ac – PP2Ac regulates Mid1 activity via dephosphorylation, and conversely, Mid1 regulates PP2Ac activity via ubiquitination. However, the precise mechanism by which alpha4 modulates these processes remains unclear.
Here, we report experimental evidence to verify the role of alpha4 as an adaptor protein that facilitates formation of a Mid1•alpha4•PP2Ac ternary complex. Interestingly, we also demonstrate that alpha4 interacts with ubiquitin and possesses a ubiquitin-interacting motif (UIM). Finally, we show that wild-type alpha4, but not an alpha4 UIM deletion mutant, suppresses PP2Ac polyubiquitination. Together, these studies reveal that alpha4 serves as an adaptor protein to directly regulate PP2Ac ubiquitination via its UIM domain.