We identified an essential Saccharomyces cerevisiae protein, Tap42, that associates with Sit4, a type 2A-related protein phosphatase, and with the type 2A phosphatase catalytic subunits. The association of Tap42 with the phosphatases does not require the previously identified phosphatase subunits. Genetic analysis suggests that Tap42 functions positively with both phosphatases. Mutations in TAP42 can confer almost complete rapamycin resistance. In addition, Tap42/Sit4 and Tap42/PP2A complex formation is regulated by nutrient growth signals and the rapamycin-sensitive Tor signaling pathway. These findings, combined with the defect in translation of the tap42-11 mutant at the nonpermissive temperature, suggest that Tap42, Sit4, and PP2A are components of the Tot signaling pathway.

Saccharomyces cerevisiae strains containing temperature-sensitive mutations in the SIT4 protein phosphatase arrest in late G1 at the nonpermissive temperature. Order-of-function analysis shows that SIT4 is required in late G1 for progression into S phase. While the levels of SIT4 do not change in the cell cycle, SIT4 associates with two high-molecular-weight phosphoproteins in a cell-cycle-dependent fashion. In addition, we have identified a polymorphic gene, SSD1, that in some versions can suppress the lethality due to a deletion of SIT4 and can also partially suppress the phenotypic defects due to a null mutation in BCY1. The SSD1 protein is implicated in G1 control and has a region of similarity to the dis3 protein of Schizosaccharomyces pombe. We have also identified a gene, PPH2alpha, that in high copy number can partially suppress the growth defect of sit4 strains. The PPH2 alpha gene encodes a predicted protein that is 80% identical to the catalytic domain of mammalian type 2A protein phosphatases but also has an acidic amino-terminal extension not present in other phosphatases.

In Saccharomyces cerevisiae, the rapamycin-sensitive TOR kinases negatively regulate the type 2A-related phosphatase SIT4 by promoting the association of this phosphatase with the inhibitor TAP42. Here, we describe TIP41, a conserved TAP42-interacting protein involved in the regulation of SIT4. Deletion of the TIP41 gene confers rapamycin resistance, suppresses a tap42 mutation, and prevents dissociation of SIT4 from TAP42. Furthermore, a TIP41 deletion prevents SIT4-dependent events such as dephosphorylation of the kinase NPR1 and nuclear translocation of the transcription factor GLN3. Thus, TIP41 negatively regulates the TOR pathway by binding and inhibiting TAP42. The binding of TIP41 to TAP42 is stimulated upon rapamycin treatment via SIT4-dependent dephosphorylation of TIP41, suggesting that TIP41 is part of a feedback loop that rapidly amplifies SIT4 phosphatase activity under TOR-inactivating conditions.

Cancer cells bypass normal controls over mitotic cell-cycle progression to achieve a deregulated state of proliferation. The retinoblastoma tumor suppressor protein (pRb) governs a key cell-cycle checkpoint that normally prevents G 1 -phase cells from entering S-phase in the absence of appropriate mitogenic signals. Cancer cells frequently overcome pRbdependent growth suppression via constitutive phosphorylation and inactivation of pRb function by cyclin-dependent kinase (CDK) 4 or CDK6 partnered with D-type cyclins.

SIT4 is the catalytic subunit of a type 2A-related protein phosphatase in Saccharomyces cerevisiae that is required for G1 cyclin transcription and for bud formation. SIT4 associates with several high-molecular-mass proteins in a cell cycle-dependent fashion. We purified two SIT4-associated proteins, SAP155 and SAP190, and cloned the corresponding genes. By sequence homology, we isolated two additional SAP genes, SAP185 and SAP4. Through such an association is not yet proven for SAP4, each of SAP155, SAP185, and SAP190 physically associates with SIT4 in separate complexes. The SAPs function positively with SIT4, and by several criteria, the loss of all four SAPs is equivalent to the loss of SIT4. The data suggest that the SAPs are not functional in the absence of SIT4 and likewise that SIT4 is not functional in the absence of the SAPs. The SAPs are hyperphoshorylated in cells lacking SIT4, raising the possibility that the SAPs are substrates of SIT4. By sequence similarity, the SAPs fall into two groups, the SAP4/SAP155 group and the SAP185/SAP190 group. Overexpression of a SAP from one group does not suppress the defects due to the loss of the other group. These findings and others indicate that the SAPs have distinct functions.