Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2005 Apr;16(4):2119-27.
doi: 10.1091/mbc.e04-09-0797. Epub 2005 Feb 2.

The yeast phosphotyrosyl phosphatase activator is part of the Tap42-phosphatase complexes

Yin Zheng  1 , Yu Jiang
Affiliations

The yeast phosphotyrosyl phosphatase activator is part of the Tap42-phosphatase complexes

Yin Zheng et al. Mol Biol Cell. 2005 Apr.

Abstract

Phosphotyrosyl phosphatase activator PTPA is a type 2A phosphatase regulatory protein that possesses an ability to stimulate the phosphotyrosyl phosphatase activity of PP2A in vitro. In yeast Saccharomyces cerevisiae, PTPA is encoded by two related genes, RRD1 and RRD2, whose products are 38 and 37% identical, respectively, to the mammalian PTPA. Inactivation of either gene renders yeast cells rapamycin resistant. In this study, we investigate the mechanism underling rapamycin resistance associated with inactivation of PTPA in yeast. We show that the yeast PTPA is an integral part of the Tap42-phosphatase complexes that act downstream of the Tor proteins, the target of rapamycin. We demonstrate a specific interaction of Rrd1 with the Tap42-Sit4 complex and that of Rrd2 with the Tap42-PP2Ac complex. A small portion of PTPA also is found to be associated with the AC dimeric core of PP2A, but the amount is significantly less than that associated with the Tap42-containing complexes. In addition, our results show that the association of PTPA with Tap42-phosphatase complexes is rapamycin sensitive, and importantly, that rapamycin treatment results in release of the PTPA-phosphatase dimer as a functional phosphatase unit.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Interaction of the Rrd proteins with Sit4 and Pph21. Yeast cells (Y162) expressing HA-PPH21 were transformed with pRS314 (lane 1), pRS314-RRD1(myc)13 (lane 2), or pRS314-RRD2(myc)13 (lane 3). (A) Interaction of the myc-tagged Rrd proteins (Rrd-myc) with Sit4 and the triple HA epitope-tagged Pph21 (HA-Pph21) was examined by coimmunoprecipitation by using anti-myc antibody (9E10). Sit4 and HA-Pph21 copurified with either Rrd1-myc (lane 2) or Rrd2-myc (lane 3) were shown by Western blotting with anti-Sit4 and -HA (12CA5) antibodies. (B) Expression levels of the Rrd proteins Sit4 and HA-Pph21 in the cell extracts used in A were analyzed by Western blotting.
Figure 2.
Figure 2.
Rrd1 and Rrd2 physically associate with Tap42. The interaction of Tap42 with either Rrd1-myc or Rrd2-myc was examined by coimmunoprecipitation. (A) Anti-Tap42 antibody was used to precipitate extracts from cells expressing a control vector (Y847, lane1), pRS314-RRD1 (myc)13 (Y846, lane2), and pRS314-RRD2(myc)13 (Y848, lane3). The presence of the myc-tagged Rrdproteins (Rrd-myc) in the precipitates was determined by Western blotting. (B) Anti-myc (9E10) antibody was used to precipitate myc-tagged Rrd1 and Rrd2 from the same cell extracts used in A.The presence of Tap42 in the myc precipitates was shown by Western blotting.
Figure 3.
Figure 3.
Rrd1 forms a ternary complex with Tap42 and Sit4. Extracts from cells expressing the RRD1(myc)13 gene (Y850) together with either a GST (lane 1) or GST-SIT4 gene (lane 2) were precipitated with glutathione beads. The presence of Rrd1-myc and Tap42 in the GST (lane 3) or GST-Sit4 (lane 4) precipitates was detected by Western blotting. Proteins released from the beads were then immunoprecipitated with anti-Tap42 antibody, and the presence of GST-Sit4 and Rrd1-myc in the Tap42 precipitates was determined by Western blotting (lanes 5 and 6).
Figure 4.
Figure 4.
Tap42 is able to interact with the Rrd proteins independent of the phosphatases. Plasmids pRS314-RRD1(myc)13 or pRS314-RRD2(myc)13 were introduced into the sit4 (Y397), pph21 pph22 (Y531) mutants as well as their isogenic wild-type alleles (Y062 and Y661). Extracts made from these strains were immunoprecipitated with anti-myc antibody. (A) Amounts of Tap42 copurified with Rrd1-myc from the wild-type (lane 1) and sit4 (lane 2) cell extracts were examined by Western blotting. Similarly, the levels of Tap42 copurified with Rrd2 from the wild-type (lane 3) and pph21 pph22 (lane 4) cell extracts were analyzed by Western blotting. (B) The levels of Tap42 in extracts of wild-type (lanes 1 and 3), sit4 (lane 2), and pph21 pph22 (lane 4) cells were examined by Western blotting. (C) Beads coated with GST (lane 1) and GST-Rrd1 (lane 2) were incubated with extracts from cells expressing Tap42 (lane 3). Tap42 bound to GST (lanes 4) and GST-Rrd1 (lane 5) was detected by Western blotting.
Figure 5.
Figure 5.
The Rrd proteins interact with the A subunit of PP2A. Extracts from cells expressing either RRD1(myc)13 (Y929) or RRD2(myc)13 (Y930) were precipitated with anti-myc antibody. The presence of Tpd3 (middle) and Cdc55-HA (bottom) in the cell extracts (Ext) and the myc precipitates (IP) was analyzed by Western blotting.
Figure 6.
Figure 6.
The Tap42–phosphatase complexes are the major targets of the Rrd proteins. Extracts from cells expressing RRD2(myc)13 (Y852) were precipitated with either anti-Tap42 or anti-Tpd3 antibody. (A) Levels of Tap42 (top) and Tpd3 (bottom) in the extracts (ext), supernatants (sup), and precipitates (IP) were analyzed by Western blotting. (B) Amounts of Rrd2-myc copurified with Tap42 and Tpd3 were compared by Western blot analysis. (C) Quantitative presentation of the Western blotting result shown in B. (D) Extracts from cells expressing RRD1(myc)13 (Y850) were precipitated with either anti-Tap42 or anti-Tpd3 antibody. The amount of Rrd1-myc copurified with either Tap42 (lane 1) or Tpd3 (lane 2) was analyzed by Western blotting.
Figure 7.
Figure 7.
Interaction between Tap42 and the Rrd proteins is rapamycin sensitive. Yeast cells (Y162) expressing either RRD1(myc)13 or RRD2(myc)13 were treated with rapamycin (200 nM) or drug vehicle for 1 h. Tap42, Sit4 and HA-Pph21 were precipitated from extracts made from the treated or untreated cells with anti-Tap42 (A and B), -Sit4 (C), and -HA (D) antibodies. (A) Western blot analysis of Rrd1-myc (middle) and Sit4 (bottom) copurified with Tap42 (top). (B) Western blot analysis of Rrd2-myc (middle) and HA-Pph21 (bottom) copurified with Tap42 (top). (C) Western blot analysis of Rrd1-myc (bottom) copurified with Sit4 (top). (D) Western blot analysis of Rrd2-myc (bottom) copurified with HA-Pph21 (top).
Figure 8.
Figure 8.
Rrd proteins are required for rapamycin-induced activation of the Sit4 phosphatase. Wild-type (Y661), rrd1 (842), rrd2 (Y843), rrd1 rrd2 (Y874), and sit4 (Y397) cells expressing a myctagged GLN3 gene were treated with or without rapamycin (200 nM) for 30 min. Cell extracts were subjected to SDS-PAGE followed by Western blotting with anti-myc antibody. The dephosphorylation of Gln3 is shown by the faster migrating bands on the SDS-PAG.
Figure 9.
Figure 9.
Rapamycin resistance of the rrd1 and rrd2 mutants is dependent upon Sit4. (A) Mid-log phase cells of wild-type (Y062), rrd1 (Y884), rrd2 (Y885), rrd1 rrd2 (Y871), sit4 (Y397), rrd1 sit4 (Y886), and rrd2 sit4 (Y887) strains were subjected to a series of 10-fold dilutions. The cells were spotted on YPD plates containing either 100 nM of rapamycin (YPD + Rap) or drug vehicle alone (YPD). Plates were photographed after incubated at 30°C for 48 h. (B) Rapamycin resistance of the wild-type (Y062), gln3 (Y275), sit4 (Y397), and gln3 sit4 (Y964) strains was assayed as described in A.
Figure 10.
Figure 10.
Rrd proteins are part of the Tap42–phosphatase complexes. The Rrd proteins exist mainly in the Tap42-containing complexes, including the Tap42–Sit4–Rrd1 and Tap42–PP2Ac–Rrd2 complexes. Rapamycin causes release of the phosphatase-Rrd dimer from Tap42.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Beck, T., and Hall, M. N. (1999). The TOR signalling pathway controls nuclear localization of nutrient-regulated transcription factors. Nature 402, 689-692. - PubMed
    1. Bertram, P. G., Choi, J. H., Carvalho, J., Ai, W., Zeng, C., Chan, T. F., and Zheng, X. F. (2000). Tripartite regulation of Gln3p by TOR, Ure2p and phosphatases. J. Biol. Chem. 275, 35727-35733. - PubMed
    1. Cardenas, M. E., Cutler, N. S., Lorenz, M. C., Di Como, C. J., and Heitman, J. (1999). The TOR signaling cascade regulates gene expression in response to nutrients. Genes Dev. 13, 3271-3279. - PMC - PubMed
    1. Cayla, X., Goris, J., Hermann, J., Hendrix, P., Ozon, R., and Merlevede, W. (1990). Isolation and characterization of a tyrosyl phosphatase activator from rabbit skeletal muscle and Xenopus laevis oocytes. Biochemistry 29, 658-667. - PubMed
    1. Cayla, X., Van Hoof, C., Bosch, M., Waelkens, E., Vandekerckhove, J., Peeters, B., Merlevede, W., and Goris, J. (1994). Molecular cloning, expression, and characterization of PTPA, a protein that activates the tyrosyl phosphatase activity of protein phosphatase 2A. J. Biol. Chem. 269, 15668-15675. - PubMed

Publication types

MeSH terms

Substances