Here we identify a critically important functional and physical interaction between RRD2 and the structural subunit TPD3 in C subunit maturation. RRD2/TPD3 interacts preferentially with the demethylated and inactive form of the C subunit by targeting the PPH21:PPE1 complex. PPE1, therefore, may fulfill a surveillance function of TPD3/RRD2-dependent C subunit maturation by preventing untimely C subunit methylation and hence the premature generation of active C subunit and holoenzyme assembly (see model, ).
Model: RRD/PTPA-Dependent Generation of Active PP2A C Subunit Is Coupled to Holoenzyme Assembly and Regulated by Methyl-Esterase/Transferase Enzymes
The catalytic subunit of a multisubunit enzyme like PP2A achieves substrate specificity by complex formation with regulatory subunits [22
]. The combinatorial assembly of holoenzymes from a large pool of distinct regulatory subunits generates a great number of different substrate-specific complexes and explains how a single catalytic subunit is able to perform so many functions. Inherent to the multisubunit enzyme architecture, however, is the problem of how the promiscuous catalytic activity of the free catalytic subunit is kept in check until holoenzymes form. Recently, we obtained experimental evidence for a potential control mechanism, which involves synthesis of the PP2A catalytic subunit in a low-activity form that requires the functional interaction with RRD/PTPA for the switch into an active and specific enzyme [13
]. Here we show that the scaffolding subunit TPD3 functionally and physically interacts with RRD2 in the generation of the active and specific PP2A C subunit, indicating that C subunit maturation is coupled and coordinated with holoenzyme assembly.
Biochemical analyses indicated that in vitro, binding of the A subunit to the C subunit decreases the phosphatase catalytic rate and increases substrate specificity [7
]. However, the effects of PP2A A subunit binding on C subunit catalytic properties were determined with C subunits isolated from catalytically active PP2A holoenzymes, because the free C subunit does not seem to exist in substantial amounts in vivo. On the basis of these in vitro observations, it has been assumed that loss of the A subunit in vivo will leave behind the monomeric C subunit with high and promiscuous catalytic activity. The data presented here demonstrate that this assumption is incorrect. Our analysis of the tpd3
Δ strain indicates that the A subunit provides an additional function besides narrowing the substrate specificity of an otherwise promiscuous C subunit. As evidenced by genetic and physical interactions, the A subunit also cooperates with RRD/PTPA in the generation of the active C subunit in vivo.
What is the regulatory role of TPD3 in this process? TPD3 formed a multimeric complex with RRD2 and PP2A C subunits. This complex appears to be essential for PP2A biogenesis and differs in several aspects from B-type subunit-containing holoenzymes. In contrast to B-type subunits, RRD2 interacted preferentially with the unmethylated C subunit, suggesting methylation state-dependent regulation of RRD2:C subunit interaction. We found, consistent with this hypothesis, that deletion of PPE1 (as well as PPM1 overexpression; unpublished data) decreased the levels of RRD2:TPD3:C subunit complexes. RRD2:TPD3:PPH21 holoenzyme assembly constitutes the first example of a PP2A complex whose stability is regulated in an opposite manner to other PP2A holoenzymes, namely by demethylation rather than methylation. The binding preference of RRD2 for the demethylated C subunit suggests an important role for PPE1, the enzyme catalyzing the demethylation, as well as for the carboxyl terminus itself in the interaction with RRD2. In accordance with our prediction, a C subunit mutant lacking the carboxy-terminal nine amino acids including the site of methylation did not bind RRD2, and the monoclonal antibody 7A6 directed to the C subunit carboxyl terminus did not co-precipitate RRD2 (unpublished data), indicating that the C subunit carboxyl terminus seems to be essential for the interaction (directly or indirectly) with RRD2. The impaired catalytic activity of the deletion mutant towards phosphorylase a (unpublished data) provided further evidence for the functional cooperation between the C subunit carboxyl terminus and RRD2.
So far, no obvious phenotype has been identified following deletion of PPE1
in yeast [10
], raising questions about PPE1′s role and importance in vivo as the opposing enzyme of the PP2A methyltransferase, PPM1. Whereas PPM1 function (C subunit methylation) is a prerequisite for stable complex formation with B-type subunits [9
], the PPE1 function in the regulation of trimeric PP2A holoenzymes remained obscure because conflicting data exist on its ability to demethylate the C subunit once it is bound to B-type subunits [23
]. Thus, it is currently thought that PPE1 regulates PP2A holoenzyme assembly with B-type subunits by shifting the equilibrium between the demethylated and the methylated AC core dimer. Our data indicate a role for PPE1 earlier in the biogenesis of PP2A, namely in the regulation of complex assembly with RRD2 rather than with B-type subunits. PPE1 appears to be required for stable complex formation between the C subunit and TPD3:RRD2, and counteracts the premature methylation of the C subunit that has not yet been activated by TPD3:RRD/PTPA. Defects in C subunit biogenesis, for example by deletion of either the TPD3
genes, caused the accumulation of complexes of PPE1 and the C subunit in its low-activity conformation. Thus, PPE1 seems to constitute a surveillance mechanism that prevents the premature generation of the active C subunit in the absence of the scaffolding subunit TPD3, the major constituent of PP2A holoenzymes, or premature holoenzyme assembly in the absence of the essential activator RRD/PTPA. PPE1 seems to achieve this function in two ways, by competing with RRD2 for the binding to the C subunit and, more importantly, by keeping the C subunit in its demethylated state and thereby preventing untimely methylation that would otherwise complete the C subunit activation and promote complex formation with B-type subunits. In line with this prediction, deletion of PPE1
in a tpd3
Δ strain overcame the PPE1-dependent block of C subunit maturation despite the inability for holoenzyme formation, and deletion of PPE1
in the rrd1
Δ strain increased dramatically the levels of trimeric holoenzymes with CDC55 despite the fact that the C subunit was in its inactive conformation.
How do defects in C subunit biogenesis lead to the increased interaction between PPE1 and the C subunit? At the moment, our data do not allow us to distinguish between several potential scenarios of PPE1 function. In the first scenario, PPE1 constitutes one of the first steps of C subunit biogenesis, interacting with the newly synthesized C subunit in its low-activity conformation. Interruptions of the biogenesis cascade would then cause the accumulation of complexes from earlier steps because dissociation of these complexes is interlocked with formation of the subsequent complex, e.g., the TPD3:RRD2 complex. In the second scenario, PPE1 plays a more active role by interacting with and demethylating C subunits that have not yet been activated by RRD/PTPA. PPE1 could achieve this by a substrate preference for any C subunit in its low-activity conformation, including those that are newly synthesized and inactive. Our results from PPE1 overexpression experiments (but also the finding of increased PPE1 binding levels in the tpd3Δ and rrd1Δ/rrd2Δ strains) are also consistent with a third mechanism in which PPE1 targets the catalytically active C subunit, demethylates it, and through its binding to catalytic site residues, converts the C subunit into the low-activity conformation, which subsequently requires RRD/PTPA as well as C subunit methylation for recycling in the biogenesis cascade.
This biogenesis mechanism appears to function in mammals as well as yeast. Consistent with an affinity of PPE1 for the inactive C subunit, we were able to identify and isolate the mammalian PPE1 ortholog, PME1, by its increased interaction with catalytically defective C subunit mutants [25
], and PME-1 was recently co-purified with inactive PP2A [24
]. Interestingly, several of the human C subunit active-site mutant proteins also bind increased levels of PTPA (the mammalian ortholog of RRD2) as well as α4/TAP42 [13
], suggesting a binding preference of both PTPA and α4/TAP42 for the inactive conformation of the C subunit. The increased binding of these molecules to C subunit mutants that are unable to fold into the catalytically active conformation and complete the biogenesis suggests that they might play key roles in the early steps of C subunit biogenesis.
TAP42 is an essential protein [17
] known to interact with the catalytic subunits of the PP2A family of phosphatases, including PPH21, PPH22, SIT4, and PPH3 [26
]. A recent study identified RRD1 and RRD2 as part of the TAP42:phosphatase complexes [20
] in the W303 strain, and confirmed earlier findings that RRD/PTPA proteins are required for phosphatase activity [13
]. In addition, these authors detected a complex between the RRD proteins and TPD3, but the significance of this complex remained unclear. Our results now identify the functional and physical interaction between RRD2 and TPD3 in C subunit biogenesis. In contrast to their findings, we were unable to detect significant complex formation between RRD2 and TAP42 in the BY strain. Thus, the observed differences may be due to the different genetic backgrounds of the W303 and BY strains. It will be interesting to test whether and how the differences in TAP42:RRD complexes affect PP2A biogenesis in these strains.
By what mechanism might RRD/PTPA regulate C subunit biogenesis? We showed recently that loss of RRD proteins results in the generation of PP2A and SIT4 catalytic subunits with impaired catalytic activity, indicating alteration of the active site. Thus, we hypothesized that RRD/PTPA might play a role in the generation of the native conformation of the PP2A and SIT4 active sites. In line with our assumption, preliminary evidence has been provided for a potential peptidyl-prolyl cis/trans
isomerase (PPIase) activity of RRD/PTPA [28
] that is stimulated by ATP/Mg2+
and, which could be responsible for switching the C subunit into the active conformation. Evidence for a potential PPIase activity was further provided by the recent structure determination of RRD/PTPA in complex with a proline-containing PPIase peptide substrate [29
]. No ATP binding site, however, could be identified in the RRD/PTPA structure [29
]. Interestingly, SSB2, a ribosome-associated chaperone that possesses ATPase activity and plays a role in the folding of newly synthesized proteins has been found just recently in a complex with TPD3 [31
]. It is tempting to speculate that the energy necessary for the PTPA-catalyzed conformational change in the C subunit might be provided by ATP hydrolysis through the TPD3-bound SSB2. It would also provide a potential explanation for our finding of the functional and physical interaction between RRD/PTPA and TPD3. Chao et al. [32
], however, identified an ATP binding pocket in their structure of PTPA and showed that PTPA and the PP2A A-C dimer together constitute a composite ATPase, whose activity is required for the transient change of PP2A into a phosphotyrosine (P-Tyr)-specific phosphatase, at least in vitro. How the loss of this transient P-Tyr–specific activity would lead to the huge decrease of the P-Ser/P-Thr–specific activity of trimeric PP2A holoenzymes that is observed in the rrd1
Δ strain [13
], or now in the rrd1
Δ strain, is unclear. The requirement for an activator protein, however, is not unique to the PP2A family of phosphatases. Inhibitor-2 (I-2), a highly conserved protein, interacts with the catalytic subunit of protein phosphatase 1 (PP1) and inhibits its catalytic activity in vitro [33
]. Deletion of GLC8,
encoding the yeast I-2 ortholog, however, leads to a reduction and not to an increase of GLC7 catalytic activity, as would have been expected for the loss of an inhibitor [34
]. The catalytically inactive PP1 in complex with I-2 is activated upon phosphorylation of I-2, which probably induces a conformational change in PP1, suggesting a function of inhibitor-2 as a PP1 chaperone [35
]. Whether the I-2 chaperone function, and thus the generation of active PP1, is also coupled to and coordinated with PP1 holoenzyme assembly is currently unknown, but—given our present results—this is a likely possibility.
Deletion of RRD/PTPA affected the post-translational modification state of the yeast B-type subunits CDC55 [13
] and RTS1 (unpublished data), suggesting the intriguing possibility that phosphorylated B-type subunits are substrates of RRD/PTPA complexes in vivo. RRD/PTPA may represent a unique species of PP2A regulatory subunits, with a very restricted number of (potentially even P-Tyr) phosphorylated substrates. In this way, the generation of the active C subunit would be tightly coupled to the formation of substrate-specific holoenzymes. Cells could avoid potential damage from the highly active but untargeted C subunit by coupling the RRD/PTPA-dependent maturation process to the interaction of the C subunit with the scaffolding subunit TPD3, which in turn constitutes a prerequisite for holoenzyme assembly with B-type subunits (model, ). The order of events seems to be under the surveillance of the PP2A methylesterase that preferentially binds to the C subunit in its low-activity conformation, consistent with PPE1′s increased affinity for inactive C subunit mutants. Demethylation of the C subunit by PPE1 is required for the efficient interaction with RRD2, and at the same time, demethylation prevents interaction with B-type subunits. TPD3 and RRD2 are both required for the release of PPE1 and the generation of the active and specific C subunit. The RRD2-dependent activation of the phosphatase catalytic activity is coupled to the assembly with B-type subunits by targeting the RRD2:TPD3:C subunit complexes to their potential substrates, phosphorylated B-type subunits. The process is completed by C subunit methylation destabilizing the interaction with RRD2 and, at the same time, stabilizing the one with B-type subunits. This model provides an explanation for how cells deal with the inherent danger of the multisubunit enzyme architecture, namely the untargeted and unrestricted catalytic activity of the free C subunit.