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. 2001 Jun 29;276(26):24253-60.
doi: 10.1074/jbc.M102398200. Epub 2001 Apr 23.

A mammalian homolog of yeast MOB1 is both a member and a putative substrate of striatin family-protein phosphatase 2A complexes

C S Moreno  1 , W S Lane, D C Pallas
Affiliations

A mammalian homolog of yeast MOB1 is both a member and a putative substrate of striatin family-protein phosphatase 2A complexes

C S Moreno et al. J Biol Chem. .

Abstract

Striatin and S/G(2) nuclear autoantigen (SG2NA) are related proteins that contain membrane binding domains and associate with protein phosphatase 2A (PP2A) and many additional proteins that may be PP2A regulatory _targets. Here we identify a major member of these complexes as class II mMOB1, a mammalian homolog of the yeast protein MOB1, and show that its phosphorylation appears to be regulated by PP2A. Yeast MOB1 is critical for cytoskeletal reorganization during cytokinesis and exit from mitosis. We show that mMOB1 associated with PP2A is not detectably phosphorylated in asynchronous murine fibroblasts. However, treatment with the PP2A inhibitor okadaic acid induces phosphorylation of PP2A-associated mMOB1 on serine. Moreover, specific inhibition of PP2A also results in hyperphosphorylation of striatin, SG2NA, and three unidentified proteins, suggesting that these proteins may also be regulated by PP2A. Indirect immunofluorescence produced highly similar staining patterns for striatin, SG2NA, and mMOB1, with the highest concentrations for each protein adjacent to the nuclear membrane. We also present evidence that these complexes may interact with each other. These data are consistent with a model in which PP2A may regulate mMOB1, striatin, and SG2NA to modulate changes in the cytoskeleton or interactions between the cytoskeleton and membrane structures.

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Figures

Fig. 1
Fig. 1. SG2NA-PP2A and striatin-PP2A form stable complexes with mammalian class II MOB1
A, amino acid sequence alignment of class II MOB1 from mouse/human, Drosophila, and C. elegans with S. cerevisiae and S. pombe MOB1. Human and mouse MOB1 are 100% identical at the amino acid level. Amino acids matching the consensus sequence are boxed. B, a whole cell lysate and the indicated immunoprecipitations (IP) were prepared from NIH3T3 cells that stably express HA-tagged mMOB1. Immune complexes were analyzed by SDS-PAGE, transferred to nitrocellulose, and sequentially probed by immunoblotting. Striatin, SG2NA, PP2A C subunit, HA-mMOB1, and endogenous (Endog.) mMOB1 were detected in 12CA5 immunoprecipitations of HA-mMOB1 but not in a control immunoprecipitation. Also, HA-mMOB1 and endogenous mMOB1 were detected in PP2A (1d6), striatin, and SG2NA immunoprecipitations but not in control immunoprecipitations. Control immunoprecipitations were performed using the 7-34-1 monoclonal antibody (American Type Culture Collection) directed against major histocompatibility complex class I swine leukocyte antigen. C, as an additional control, some lysates were preboiled in 0.5% SDS and 5 mm β-mercaptoethanol to disrupt complexes, and then parallel immunoprecipitations were prepared. The absence of co-immunoprecipitating proteins from preboiled lysates indicates that co-immunoprecipitations were not due to cross-reactive antibodies.
Fig. 2
Fig. 2. Mammalian MOB1 is covalently modified in vivo
NIH3T3 cells stably expressing HA-mMOB1 were metabolically labeled with [35S]methionine, and SG2NA and control immunoprecipitations (IP) were analyzed by two-dimensional gel electrophoresis. Isoelectric focusing was from right (basic end) to left (acidic end). Control immunoprecipitations were performed with preimmune sera from the same rabbit used to generate SG2NA antibodies. Both endogenous mMOB1 and HA-tagged mMOB1 and PP2A A subunit (A sub) and C subunit (C sub) are indicated. Also shown are the locations of unidentified members of the SG2NA-PP2A-MOB1 complexes that migrate at 47, 52, and 60 kDa, respectively. The multiple spots indicated by the SG2NA bracket (*) include SG2NA and striatin as well as possibly other striatin-SG2NA family members or alternatively spliced forms of SG2NA.
Fig. 3
Fig. 3. PP2A inhibition in vivo by okadaic acid treatment results in phosphorylation of mMOB1 and HA-mMOB1 and hyperphosphorylation of SG2NA and three unidentified proteins of 47, 52, and 60 kDa
NIH3T3 cells that stably express HA-mMOB1 were metabolically labeled with 32P and treated with 1 μm okadaic acid or left as untreated controls. Immunoprecipitations (IP) using preimmune antisera and anti-SG2NA antisera were prepared from radiolabeled cells and analyzed on SDS-PAGE, and phosphorylated proteins were detected by autoradiography. HA-mMOB1 and endogenous mMOB1 can be detected only in cells treated with okadaic acid. The migration positions of the various 35S-labeled proteins shown in Fig. 2 are indicated by brackets and arrows.
Fig. 4
Fig. 4. mMOB1 is phosphorylated only on serine residues, whereas SG2NA and p52 are phosphorylated on both serine and threonine
NIH3T3 cells were labeled in vivo with [32P]orthophosphate and either treated (1) with 1 mm okadaic acid or left untreated (–). SG2NA complexes were immunoprecipitated and analyzed by SDS-PAGE, and phosphoamino acid analysis was performed as described (38). In the absence of okadaic acid, mMOB1 and p52 cannot be detected with 32P and, thus, are not shown. Phosphoserine residues (S) and phosphothreonine residues (T) are indicated on the left of the figure. No phosphotyrosine was detected in any of these proteins (not shown).
Fig. 5
Fig. 5. SG2NA and striatin are substrates of PP2A
NIH3T3 cells were treated with increasing concentrations of okadaic acid, and HA-mMOB1 complexes were immunoprecipitated (IP) and immunoblotted. Phosphorylation of SG2NA can be detected by a slower migration of SG2NA on SDS-PAGE. Partial phosphorylation of SG2NA can be detected with okadaic acid concentrations as low as 100 nm, and complete phosphorylation is observed at 200 nm, indicating that SG2NA may be a PP2A substrate. Striatin phosphorylation can also be detected at 100 nm okadaic acid. The migration of mMOB1 on SDS-PAGE is unaffected by phosphorylation.
Fig. 6
Fig. 6. A subpopulation of striatin, SG2NA, and HA-mMOB1 appear to colocalize around the nuclear periphery
Murine fibroblasts were fixed in 2% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and stained for the presence of HA-mMOB1 (panels A, D, G, J, and M), SG2NA (panels B, E, H, and P), or striatin (panels K, N, and Q). Each set of three panels indicates the fluorescence observed using mouse monoclonal antibodies shown in green, rabbit polyclonal antibodies shown in red, and a third panel indicating the merge of the two signals. Panels A, B, and C are co-stained with 16b12 and affinity-purified SG2NA antibodies. Panels D, E, and F show the fluorescence observed in HA-mMOB1 cells co-stained with the 16b12 antibody and affinity-purified SG2NA antibodies preincubated with 2 μg/ml SG2NA peptide. The lack of signal in panel E and the presence of a HA-mMOB1 signal in panel D indicate that the SG2NA signal is specific. All panels used HA-mMOB1 cells except panels G, H, and I, which used the parental NIH3T3 cell line, demonstrating the specificity of the HA-mMOB1 signal. Panels J, K, and L are co-stained with 16b12 and affinity-purified striatin antibodies. In panels M, N, and O, the 16b12 and striatin antibodies were preincubated with 2 μg/ml striatin peptide. Panels P, Q, and R are co-stained with a monoclonal antibody to SG2NA (green) and affinity-purified striatin antibodies (red).
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