Human Protein Phosphatase PP6 Regulatory Subunits Provide Sit4-Dependent and Rapamycin–Sensitive Sap Function in Saccharomyces cerevisiae

Abstract

In the budding yeast Saccharomyces cerevisiae the protein phosphatase Sit4 and four associated proteins (Sap4, Sap155, Sap185, and Sap190) mediate G1 to S cell cycle progression and a number of signaling events controlled by the target of rapamycin TOR signaling cascade. Sit4 and the Sap proteins are ubiquitously conserved and their human orthologs, PP6 and three PP6R proteins, share significant sequence identity with their yeast counterparts. However, relatively little is known about the functions of the PP6 and PP6R proteins in mammalian cells. Here we demonstrate that the human PP6R proteins physically interact with Sit4 when expressed in yeast cells. Remarkably, expression of PP6R2 and PP6R3 but not expression of PP6R1 rescues the growth defect and rapamycin hypersensitivity of yeast cells lacking all four Saps, and these effects require Sit4. Moreover, PP6R2 and PP6R3 enhance cyclin G1 gene expression and DNA synthesis, and partially abrogate the G1 cell cycle delay and the budding defect of the yeast quadruple sap mutant strain. In contrast, the human PP6R proteins only modestly support nitrogen catabolite gene expression and are unable to restore normal levels of eIF2α phosphorylation in the quadruple sap mutant strain. These results illustrate that the human PP6-associated proteins are capable of providing distinct rapamycin-sensitive and Sit4-dependent Sap functions in the heterologous context of the yeast cell. We hypothesize that the human Saps may play analogous roles in mTORC1-PP6 signaling events in metazoans.

Protein Phosphatase 6 Interacts with the DNA-Dependent Protein Kinase Catalytic Subunit and Dephosphorylates γ-H2AX

“…9). Indeed, in budding yeast, the regulatory subunits SAP1, SAP2, and SAP3 have each been shown to confer different properties on Sit4, the yeast homologue of PP6c (41). We speculate that one possible function of the different PP6 complexes may be to target ␥-H2AX at foci that are located at different distances from the DSB site.…”

PP6 Regulatory Subunit R1 Is Bidentate Anchor for Targeting Protein Phosphatase-6 to DNA-dependent Protein Kinase

“…PP6 is conserved among all eukaryotic species from yeast to humans, attesting to its fundamental importance. The Saccharomyces cerevisiae homologue, Sit4, is required for G 1 to S progression, expression of G 1 cyclins, and several other cellular processes (23,26). The human PP6 also promotes G 1 to S progression, predominantly by regulating the levels of cyclin D1 (27).…”

“…In fact, R1, R2 and R3 were originally identified by the sequence homology of their SAPS domains to their yeast counterparts (31). Although the different PP6c regulatory subunits may have some redundant functions, as in the case of Aurora A dephosphorylation (28), there is evidence that each subunit may also have specific non-redundant functions (26,31). Knockdown of R1, but not R3, enhances degradation of endogenous IB⑀ in response to TNF-␣ (31).…”

Adaptation of HepG2 cells to a steady-state reduction in the content of protein phosphatase 6 (PP6) catalytic subunit

“…It has further been reported that human SAPS, when expressed in S. cerevisiae , are able to physically interact with Sit4, and that the expression of either SAPS2 or SAPS3, but not SAPS1, can rescue the growth defect and rapamycin sensitivity of yeast cells lacking all four yeast SAPs. This effect was dependent on the presence of Sit4 [12]. …”