A novel and essential mechanism determining specificity and activity of protein phosphatase 2A (PP2A) in vivo

Abstract

Protein phosphatase 2A (PP2A) is an essential intracellular serine/threonine phosphatase containing a catalytic subunit that possesses the potential to dephosphorylate promiscuously tyrosine-phosphorylated substrates in vitro. How PP2A acquires its intracellular specificity and activity for serine/threonine-phosphorylated substrates is unknown. Here we report a novel and phylogenetically conserved mechanism to generate active phospho-serine/threonine-specific PP2A in vivo. Phosphotyrosyl phosphatase activator (PTPA), a protein of so far unknown intracellular function, is required for the biogenesis of active and specific PP2A. Deletion of the yeast PTPA homologs generated a PP2A catalytic subunit with a conformation different from the wild-type enzyme, as indicated by its altered substrate specificity, reduced protein stability, and metal dependence. Complementation and RNA-interference experiments showed that PTPA fulfills an essential function conserved from yeast to man. Protein phosphorylation is a posttranslational modification, mostly reversible, that is used in cells for the regulation of multiple processes. Analyses of eukaryotic genomes reveal that the genes coding for protein kinases, the enzymes catalyzing the phosphorylation reaction, outnumber by two-to threefold genes for protein phosphatases, the enzymes catalyzing dephosphorylation (Zolnierowicz 2000). Protein phosphatases counterbalance the activity of the large number of substrate-specific kinases by the combinatorial assembly of holoenzymes with different substrate specificity. Holoenzymes of a certain protein phosphatase family consist of a common catalytic subunit associated with different regulatory subunits that determine substrate targeting and modulate catalytic activity. Hence, the catalytic subunits of the major protein phosphatase families are produced in abundance. For instance, the catalytic subunit (C subunit) of protein phosphatase 2A (PP2A), comprises, dependent on the cell type, 0.3%-1% of total cellular protein (Virshup 2000).Based on its specificity for phosphorylated serine/ threonine residues, the PP2A C subunit belongs to the family of eukaryotic protein-serine/threonine phosphatases (PSTPs). PSTPs possess a catalytic core structure that is distinct from the core of protein tyrosine phosphatases (PTPs) and dual specificity phosphatases (DSPs). In consequence of the structural differences, the different protein phosphatase families use distinct catalytic mechanisms for the hydrolysis of the phosphoester bond. In contrast to PTPs (and the DSP subfamily) PSTPs are metallo-phosphoesterases that require metals in the active site for catalysis and for their structural integrity. When isolated from eukaryotic sources, the PSTP family members protein phosphatase 1 (PP1) and PP2A ("native" PP1 or PP2A) do not require the addition of metal ions for their activity. However, PP1 and PP2A convert into metal-dependent enzymes during long-term storage or on treatment with the phosphatase inhibitors ATP, pyrophosphate (PPi), or NaF (Burche...

PP2A holoenzyme assembly: in cauda venenum (the sting is in the tail)

“…Nevertheless, PP2A can auto-dephosphorylate Tyr307 in an OA-sensitive reaction in vitro [48] and, in a YPA1/YPA2-null yeast strain, PP2A can dephosphorylate pNPP (a bulky phosphotyrosyl-mimic). This finding indicates the existence of a conformationally relaxed catalytic center that directs alterations in substrate specificity in the absence of PTPA [59]. Moreover, PTPA can activate the Ser/Thr phosphatase activity of a native inactive PP2A form isolated from tissues in complex with PME-1 [46] via a mechanism involving a peptidyl prolyl isomerase activity [57,58].…”

“…His59!Gln, His118!Gln, Asp85!Asn and Arg!88Ala) [45] or in vivo via a mechanism that remains poorly defined [46,[57][58][59][60], its interaction surface drastically changes, resulting in an increased affinity for several interacting proteins such as PME-1 [45,46], the PP2A phosphatase activator (PTPA) [45] (Box 4) and other, as yet unidentified proteins [45]. Activity of the native inactive PP2A (PP2A i ) in complex with PME-1 cannot be restored by LCMT1-mediated (re)methylation [46].…”

“…The mechanism of this activation has recently been partially solved by demonstrating that PTPA has an ATP-Mg 2+ -stimulated peptidyl prolyl cis/trans isomerase activity that targets Pro190 in PP2A C [57], which is essential for PP2A activation [58]. In a YPA1/YPA2-null yeast strain (yeast PP2A activator 1 and 2, encoding the yeast homologs of PTPA), PP2A C has decreased Ser/Thr phosphatase activity and is hypomethylated owing to its increased association with PME-1 [59,60]. The additional deletion of PPE1 (protein phosphatase methylesterase 1, encoding the yeast PME-1 homolog) in this strain restores PP2A C methylation but not Ser/Thr phosphatase activity, confirming that demethylation itself does not cause PP2A inactivation and that the inactive conformation does not prevent methylation.…”

The Crystal Structure of a Human PP2A Phosphatase Activator Reveals a Novel Fold and Highly Conserved Cleft Implicated in Protein-Protein Interactions

“…The reactivation of PP2Ac by PTPA in vitro was at first only identified as an increase in phosphotyrosyl phosphatase activity (11,12) when in fact it appears to activate the phosphoserine/threonine-specific activity of PP2Ac from a poorly active and substrate unspecific metal-dependent state (13,14). PTPA is an essential protein as revealed by its high evolutionary conservation as well as the lethality of the deletion of the two PTPA homologs in yeast, Rrd1 and Rrd2, in certain nutritional backgrounds (15).…”

Critical Role for Protein Phosphatase 2A Heterotrimers in Mammalian Cell Survival

“…Most PP2A catalytic subunits in the cell form heterotrimeric complexes followed by the AC core dimer and, finally, numerous lower abundance complexes (22)(23)(24)(25)(26)(27). Highlighting the role of PP2A in growth control, several viruses encode proteins that subvert PP2A activity by binding to either dimeric or trimeric forms of the phosphatase (1).…”