Antonioli, S. | Search bioRxiv

bioRxiv2025

A complex of MAST1 and 14-3-3 regulates Tau phosphorylation in the developing cortex

The MAST family of serine/threonine kinases has been implicated in a spectrum of human neurodevelopmental disorders. However, little is known about their biological function or regulation. Seeking to fill these gaps in our knowledge, we have identified upstream and downstream partners of MAST1. 14-3-3, a neuronal 14-3-3 paralog, specifically interacts with MAST1 at two regulatory serines, S90 and S161. PAK, a neuronal regulator of the actin cytoskeleton, phosphorylates MAST1 to regulate its interaction with 14-3-3. Exploiting mouse models of human Mega Corpus Callosum Syndrome (MCC) and whole brain phosphoproteomics, we identify the microtubule-associated protein Tau as a substrate of MAST1. We show that pathogenic MAST1 mutations perturb protein function either through misfolding or attenuation of kinase activity. Our data is consistent with a model in which the MAST kinases couple PAK, a neuronal regulator of the actin cytoskeleton, to microtubule remodeling during the differentiation and specification of cortical neurons.

molecular biology↗

bioRxiv2024

PHLPP2 is a pseudophosphatase that lost activity in the metazoan ancestor

The phosphoinositide 3-kinase (PI3K) pathway is a major regulator of cell and organismal growth. Consequently, hyperactivation of PI3K and its downstream effector kinase, Akt, is observed in many human cancers. PH domain leucine-rich repeat-containing protein phosphatases (PHLPP), two paralogous members of the metal-dependent protein phosphatase family, have been reported as negative regulators of Akt signaling and, therefore, tumor suppressors. However, the stoichiometry and identity of the bound metal ion(s), mechanism of action, and enzymatic specificity of these proteins are not known. Seeking to fill these gaps in our understanding of PHLPP biology, we unexpectedly discovered that PHLPP2 has no catalytic activity against the regulatory phosphorylation sites of Akt, nor the generic substrate para -nitrophenylphosphate. Instead, we found that PHLPP2 is a pseudophosphatase with a single zinc ion bound in its catalytic center. Furthermore, we found that current cancer genomics data do not support the proposed role of PHLPP1 or PHLPP2 as tumor suppressors. Phylogenetic analyses revealed an ancestral phosphatase that arose more than 1 Mya, but that lost activity at the base of the metazoan lineage. In summary, our results provide a molecular explanation for the inconclusive results that have hampered research on PHLPP and argue for a new focus on non-catalytic roles of PHLPP1 and PHLPP2. Significance StatementPHLPP1 and PHLPP2 have previously been reported as protein phosphatases that specifically inactivate Akt, a pro-growth and survival kinase hyperactivated in many human cancers. Unexpectedly, we found that purified PHLPP2 has no detectable enzymatic activity in vitro, an observation which can be rationalized by its unusual active site, which has diverged significantly from that of canonical metal-dependent phosphatases. Furthermore, we show that cancer genomics do not support a role for either PHLPP1 or PHLPP2 in cancer. Our findings argue for the exploration of alternative hypotheses regarding the role of PHLPP in Akt signaling and cancer, with a focus on its non-catalytic functions.

biochemistry↗