EFFECT OF JOB SKILLS TRAINING ON EMPLOYMENT AND JOB SEEKING BEHAVIORS IN AN AMERICAN INDIAN SUBSTANCE ABUSE TREATMENT SAMPLE.

Employment difficulties are common among American Indian individuals in substance abuse treatment. To address this problem, the Southwest Node of NIDA's Clinical Trials Network conducted a single-site adaptation of its national Job Seekers Workshop study in an American Indian treatment program, Na'Nizhoozhi Center (NCI). 102 (80% men, 100% American Indian) participants who were in residential treatment and currently unemployed were randomized to (1) a three session, manualized program (Job seekers workshop: JSW) or (2) a 40-minute Job Interviewing Video: JIV). Outcomes were assessed at 3-month follow up: 1) number of days to a new taxed job or enrollment in a job-training program, and 2) total hours working or enrolled in a job-training program. No significant differences were found between the two groups for time to a new taxed job or enrollment in a job-training program. There were no significant differences between groups in substance use frequency at 3-month follow-up. These results do not support the use of the costly and time-consuming JSW intervention in this population and setting. Despite of the lack of a demonstrable treatment effect, this study established the feasibility of including a rural American Indian site in a rigorous CTN trial through a community-based participatory research approach.

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

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.

Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.

Binding of different regulatory subunits and methylation of the catalytic (C) subunit carboxy-terminal leucine 309 are two important mechanisms by which protein phosphatase 2A (PP2A) can be regulated. In this study, both genetic and biochemical approaches were used to investigate regulation of regulatory subunit binding by C subunit methylation. Monoclonal antibodies selectively recognizing unmethylated C subunit were used to quantitate the methylation status of wild-type and mutant C subunits. Analysis of 13 C subunit mutants showed that both carboxy-terminal and active site residues are important for maintaining methylation in vivo. Severe impairment of methylation invariably led to a dramatic decrease in Balpha subunit binding but not of striatin, SG2NA, or polyomavirus middle tumor antigen (MT) binding. In fact, most unmethylated C subunit mutants showed enhanced binding to striatin and SG2NA. Certain carboxy-terminal mutations decreased Balpha subunit binding without greatly affecting methylation, indicating that Balpha subunit binding is not required for a high steady-state level of C subunit methylation. Demethylation of PP2A in cell lysates with recombinant PP2A methylesterase greatly decreased the amount of C subunit that could be coimmunoprecipitated via the Balpha subunit but not the amount that could be coimmunoprecipitated with Aalpha subunit or MT. When C subunit methylation levels were greatly reduced in vivo, Balpha subunits were found complexed exclusively to methylated C subunits, whereas striatin and SG2NA in the same cells bound both methylated and unmethylated C subunits. Thus, C subunit methylation is critical for assembly of PP2A heterotrimers containing Balpha subunit but not for formation of heterotrimers containing MT, striatin, or SG2NA. These findings suggest that methylation may be able to selectively regulate the association of certain regulatory subunits with the A/C heterodimer.

Carboxymethylation of the PP2A catalytic subunit in Saccharomyces cerevisiae is required for efficient interaction with the B-type subunits Cdc55p and Rts1p.

Protein phosphatase 2A (PP2A) is an essential eukaryotic serine/threonine phosphatase known to play important roles in cell cycle regulation. Association of different B-type targeting subunits with the heterodimeric core (A/C) enzyme is known to be an important mechanism of regulating PP2A activity, substrate specificity, and localization. However, how the binding of these targeting subunits to the A/C heterodimer might be regulated is unknown. We have used the budding yeast Saccharomyces cerevisiae as a model system to investigate the hypothesis that covalent modification of the C subunit (Pph21p/Pph22p) carboxyl terminus modulates PP2A complex formation. Two approaches were taken. First, S. cerevisiae cells were generated whose survival depended on the expression of different carboxyl-terminal Pph21p mutants. Second, the major S. cerevisiae methyltransferase (Ppm1p) that catalyzes the methylation of the PP2A C subunit carboxyl-terminal leucine was identified, and cells deleted for this methyltransferase were utilized for our studies. Our results demonstrate that binding of the yeast B subunit, Cdc55p, to Pph21p was disrupted by either acidic substitution of potential carboxyl-terminal phosphorylation sites on Pph21p or by deletion of the gene for Ppm1p. Loss of Cdc55p association was accompanied in each case by a large reduction in binding of the yeast A subunit, Tpd3p, to Pph21p. Moreover, decreased Cdc55p and Tpd3p binding invariably resulted in nocodazole sensitivity, a known phenotype of CDC55 or TPD3 deletion. Furthermore, loss of methylation also greatly reduced the association of another yeast B-type subunit, Rts1p. Thus, methylation of Pph21p is important for formation of PP2A trimeric and dimeric complexes, and consequently, for PP2A function. Taken together, our results indicate that methylation and phosphorylation may be mechanisms by which the cell dynamically regulates PP2A complex formation and function.

WD40 repeat proteins striatin and S/G(2) nuclear autoantigen are members of a novel family of calmodulin-binding proteins that associate with protein phosphatase 2A.

Protein phosphatase 2A (PP2A) is a multifunctional serine/threonine phosphatase that is critical to many cellular processes including development, neuronal signaling, cell cycle regulation, and viral transformation. PP2A has been implicated in Ca(2+)-dependent signaling pathways, but how PP2A is targeted to these pathways is not understood. We have identified two calmodulin (CaM)-binding proteins that form stable complexes with the PP2A A/C heterodimer and may represent a novel family of PP2A B-type subunits. These two proteins, striatin and S/G(2) nuclear autoantigen (SG2NA), are highly related WD40 repeat proteins of previously unknown function and distinct subcellular localizations. Striatin has been reported to associate with the post-synaptic densities of neurons, whereas SG2NA has been reported to be a nuclear protein expressed primarily during the S and G(2) phases of the cell cycle. We show that SG2NA, like striatin, binds to CaM in a Ca(2+)-dependent manner. In addition to CaM and PP2A, several unidentified proteins stably associate with the striatin-PP2A and SG2NA-PP2A complexes. Thus, one mechanism of targeting and organizing PP2A with components of Ca(2+)-dependent signaling pathways may be through the molecular scaffolding proteins striatin and SG2NA.

Catalytically inactive protein phosphatase 2A can bind to polyomavirus middle tumor antigen and support complex formation with pp60(c-src).

Interaction between the heterodimeric form of protein phosphatase 2A (PP2A) and polyomavirus middle T antigen (MT) is required for the subsequent assembly of a transformation-competent MT complex. To investigate the role of PP2A catalytic activity in MT complex formation, we undertook a mutational analysis of the PP2A 36-kDa catalytic C subunit. Several residues likely to be involved in the dephosphorylation mechanism were identified and mutated. The resultant catalytically inactive C subunit mutants were then analyzed for their ability to associate with a cellular (B subunit) or a viral (MT) B-type subunit. Strikingly, while all of the inactive mutants were severely impaired in their interaction with B subunit, most of these mutants formed complexes with polyomavirus MT. These findings indicate a potential role for these catalytically important residues in complex formation with cellular B subunit, but not in complex formation with MT. Transformation-competent MT is known to associate with, and modulate the activity of, several cellular proteins, including pp60(c-src) family kinases. To determine whether association of MT with an active PP2A A-C heterodimer is necessary for subsequent association with pp60(c-src), catalytically inactive C subunits were examined for their ability to form complexes containing pp60(c-src) in MT-expressing cells. Two catalytically inactive C subunit mutants that efficiently formed complexes with MT also formed complexes that included an active pp60(c-src) kinase, demonstrating that PP2A activity is not essential in cis in MT complexes for subsequent pp60(c-src) association.

A protein phosphatase methylesterase (PME-1) is one of several novel proteins stably associating with two inactive mutants of protein phosphatase 2A.

Carboxymethylation of proteins is a highly conserved means of regulation in eukaryotic cells. The protein phosphatase 2A (PP2A) catalytic (C) subunit is reversibly methylated at its carboxyl terminus by specific methyltransferase and methylesterase enzymes which have been purified, but not cloned. Carboxymethylation affects PP2A activity and varies during the cell cycle. Here, we report that substitution of glutamine for either of two putative active site histidines in the PP2A C subunit results in inactivation of PP2A and formation of stable complexes between PP2A and several cellular proteins. One of these cellular proteins, herein named protein phosphatase methylesterase-1 (PME-1), was purified and microsequenced, and its cDNA was cloned. PME-1 is conserved from yeast to human and contains a motif found in lipases having a catalytic triad-activated serine as their active site nucleophile. Bacterially expressed PME-1 demethylated PP2A C subunit in vitro, and okadaic acid, a known inhibitor of the PP2A methylesterase, inhibited this reaction. To our knowledge, PME-1 represents the first mammalian protein methylesterase to be cloned. Several lines of evidence indicate that, although there appears to be a role for C subunit carboxyl-terminal amino acids in PME-1 binding, amino acids other than those at the extreme carboxyl terminus of the C subunit also play an important role in PME-1 binding to a catalytically inactive mutant.

Reaction and movement time variability in ADD/H: effect of tactile experience.

A clinical group of children with attention deficit disorders with hyperactivity (ADD/H) was compared to a control group, each divided into high and low variability (HV and LV) on reaction time (RT) and movement time (MT) measures. The effects of tactile-somatosensory experience on performance was also investigated. Analyses of the HV and LV groups based on RT variability found improvement on MT in both HV groups (ADD/H and control) following a tactile-somatosensory task. Both HV groups based on MT variability also showed improvement in MT following the TPT administration. No LV group (based on either RT or MT) showed improvement in MT or RT. The results in this study indicated that HV (either RT or MT) predicted improved speed and accuracy following a tactile somatosensory task. Several neuroanatomical models for the study of response variability and the role of tactile somatosensory training programmes in paediatric rehabilitation are discussed.

Serine 257 phosphorylation regulates association of polyomavirus middle T antigen with 14-3-3 proteins.

Polyomavirus middle T antigen (MT) is phosphorylated on serine residues. Partial proteolytic mapping and Edman degradation identified serine 257 as a major site of phosphorylation. This was confirmed by site-directed mutagenesis. Isoelectric focusing of immunoprecipitated MT from transfected 293T cells showed that phosphorylation on wild-type MT occurred at near molar stoichiometry at S257. MT was previously shown to be associated with 14-3-3 proteins, which have been connected to cell cycle regulation and signaling. The association of 14-3-3 proteins with MT depended on the serine 257 phosphorylation site. This has been demonstrated by comparing wild-type and S257A mutant MTs expressed with transfected 293T cells or with Sf9 cells infected with recombinant baculoviruses. The 257 site is not critical for transformation of fibroblasts in vitro, since S257A and S257C mutant MTs retained the ability to form foci or colonies in agar. The tumor profile of a virus expressing S257C MT showed a striking deficiency in the induction of salivary gland tumors. The basis for this defect is uncertain. However, differences in activity for the wild type and mutant MT lacking the 14-3-3 binding site have been observed in transient reporter assays.

Protein phosphatase 2A subunit assembly: the catalytic subunit carboxy terminus is important for binding cellular B subunit but not polyomavirus middle tumor antigen.

The carboxy terminus of protein phosphatase 2A (PP2A) catalytic subunit is highly conserved. Seven out of the last nine residues, including two potential in vivo phosphorylation sites, threonine 304 and tyrosine 307, are completely invariant in all known PP2As. Mutational analysis of the carboxy terminus in vivo was facilitated by efficient immunoprecipitation of trimeric PP2A holoenzyme via an epitope-tagged catalytic subunit. The results indicate that the catalytic subunit carboxy terminus is important for complex formation with the PP2A 55 kDa regulatory B subunit, but not with polyomavirus oncogene, middle tumor antigen (MT), a viral B-type regulatory subunit. Replacing catalytic subunit threonine 304 or tyrosine 307 with a negatively charged amino acid abolished binding of the B subunit to the dimeric enzyme core and altered substrate specificity. Certain other amino acid substitutions of different size and/or charge also abolished or greatly reduced B subunit binding. Substitution of alanine at position 304 or phenylalanine at position 307 did not dramatically reduce B subunit binding or phosphatase activity in vitro, yet the latter substitutions are not found in naturally occurring PP2As. Thus, the wild-type residues are important for a yet unknown function in vivo. Additionally, deleting the carboxy terminal nine amino acids inhibited binding of the B subunit to the dimeric enzyme core, indicating a requirement for one or more of these amino acids for complex formation. MT interaction with the dimeric PP2A enzyme core was not inhibited by any of these mutations. Finally, unlike B subunit, MT does not activate the phosphatase activity of the PP2A heterodimer towards cdc2-phosphorylated histone H1.

Identification of regions in polyomavirus middle T and small t antigens important for association with protein phosphatase 2A.

Two subunits of protein phosphatase 2A (PP2A) have been shown previously to bind to the small t and middle T antigens (ST and MT, respectively) of polyomavirus. To determine sequences important for binding of PP2A to ST and MT, we first constructed a series of ST mutants in regions known to be important for biological activity of ST and MT. Several mutations in two small regions just amino terminal to the Cys-X-Cys-X-X-Cys motifs of ST and MT abolished PP2A binding to ST in vitro. Parallel mutations were constructed in MT to investigate the role of PP2A binding in the function of polyomavirus MT. Wild-type and mutant MT proteins were stably expressed in NIH 3T3 cells and analyzed (i) for their ability to induce transformation and (ii) for associated cellular proteins and corresponding enzymatic activities previously described as associating with wild-type MT. A number of the mutant MTs were found to be defective in binding of PP2A as assayed by coimmunoprecipitation. In contrast, a deletion of the highly conserved stretch of amino acids 42 to 47 (His-Pro-Asp-Lys-Gly-Gly) in the ST-MT-large T antigen common region did not affect PP2A binding to MT. MT mutants defective for PP2A binding were also defective in transformation, providing further evidence that association with PP2A is important for the ability of MT to transform cells. All mutants which were impaired for PP2A binding were similarly or more dramatically impaired for associated protein and lipid kinase activities, supporting the possibility that PP2A binding is necessary for the formation and/or stability of an MT-pp60c-src complex.

Interaction of the protein kinase Raf-1 with 14-3-3 proteins.

Members of a family of highly conserved proteins, termed 14-3-3 proteins, were found by several experimental approaches to associate with Raf-1, a central component of a key signal transduction pathway. Optimal complex formation required the amino-terminal regulatory domain of Raf-1. The association of 14-3-3 proteins and Raf-1 was not substantially affected by the activation state of Raf.

One exon of the human LSF gene includes conserved regions involved in novel DNA-binding and dimerization motifs.

The transcription factor LSF, identified as a HeLa protein that binds the simian virus 40 late promoter, recognizes direct repeats with a center-to-center spacing of 10 bp. The characterization of two human cDNAs, representing alternatively spliced mRNAs, provides insight into the unusual DNA-binding and oligomerization properties of LSF. The sequence of the full-length LSF is identical to that of the transcription factors alpha CP2 and LBP-1c and has similarity to the Drosophila transcription factor Elf-1/NTF-1. Using an epitope-counting method, we show that LSF binds DNA as a homodimer. LSF-ID, which is identical to LBP-1d, contains an in-frame internal deletion of 51 amino acids resulting from alternative mRNA splicing. Unlike LSF, LSF-ID did not bind LSF DNA-binding sites. Furthermore, LSF-ID did not affect the binding of LSF to DNA, suggesting that the two proteins do not interact. Of three short regions with a high degree of homology between LSF and Elf-1/NTF-1, LSF-ID lacks two, which are predicted to form beta-strands. Double amino acid substitutions in each of these regions eliminated specific DNA-binding activity, similarly to the LSF-ID deletion. The dimerization potential of these mutants was measured both by the ability to inhibit the binding of LSF to DNA and by direct protein-protein interaction studies. Mutations in one homology region, but not the other, functionally eliminated dimerization.

Polyoma middle tumor antigen interacts with SHC protein via the NPTY (Asn-Pro-Thr-Tyr) motif in middle tumor antigen.

Polyomavirus middle tumor antigen (MT) transforms a large number of cell types by binding to and modulating the activities of cellular proteins. Previous genetic analysis defined in MT an independent motif, NPTY (Asn-Pro-Thr-Tyr), required for transformation. This report demonstrates that NPTY is required for interaction between MT and SHC protein, a Src homology 2 (SH2)-containing protooncogene product implicated in activating Ras via association with GRB2 protein. SHC is phosphorylated on tyrosine and associates with GRB2 in MT-transformed cells. These effects require an intact NPTY motif in MT. SHC immunoprecipitates from MT-transformed cells possess kinase activity that phosphorylates not only SHC and MT but also the 85-kDa subunit of phosphatidylinositol 3-kinase. This result suggests that a complex exists that contains, at a minimum, MT, Src family tyrosine kinases, phosphatidylinositol 3-kinase, and SHC.

Association of polyomavirus middle tumor antigen with 14-3-3 proteins.

To carry out its transformation function, the middle tumor antigen (MT) of murine polyomavirus associates with a number of cellular proteins involved in regulation of cell proliferation, including pp60c-Src, phosphatidylinositol 3-kinase, protein phosphatase 2A, Src homologous and collagen protein and growth factor receptor-binding protein 2. Here, two additional MT-associated proteins were identified as members of the 14-3-3 family of proteins. Yeast homologs of 14-3-3 proteins have recently been shown to play a role in the timing of mitosis. Thus, regulation of 14-3-3 protein function by MT may contribute to the development of neoplasia.

The third subunit of protein phosphatase 2A (PP2A), a 55-kilodalton protein which is apparently substituted for by T antigens in complexes with the 36- and 63-kilodalton PP2A subunits, bears little resemblance to T antigens.

The small and middle T (tumor) antigens of polyomavirus have been shown previously to associate with the 36-kDa catalytic subunit and the 63-kDa regulatory subunit of protein phosphatase type 2A, apparently substituting for a normal third 55-kDa regulatory subunit (D.C. Pallas, L.K. Shahrik, B.L. Martin, S. Jaspers, T.B. Miller, D.L. Brautigan, and T.M. Roberts, Cell 60:167-176, 1990). To facilitate a comparison of the normal regulatory subunit and T antigens, we isolated a 2.14-kb cDNA clone encoding this 55-kDa subunit from a rat liver library. Using a probe from the coding region of this gene, we detected a major 2.4-kb mRNA transcript in liver and muscle RNAs. The 55-kDa protein phosphatase 2A subunit purified from rat skeletal muscle generates multiple species when analyzed on two-dimensional gels. Transcription and translation of the clone in vitro produced a full-length protein that comigrated precisely on two-dimensional gels with three of these species, indicating that the 55-kDa protein is apparently modified similarly in vivo and in reticulocyte lysates. Additional species in the purified preparation were not found in the translate, suggesting that there are probably two or more isoforms of this protein in rat muscle. Somewhat surprisingly, there was no clear homology with T-antigen amino acid sequences.

Increased mean serum thyrotropin in apparently euthyroid hypercholesterolemic patients: does it mean occult hypothyroidism?

Serum thyroid hormones and antithyroid autoantibodies (AAB) were assayed in 87 randomly selected hypercholesterolemic persons compared to 80 controls with normal serum total cholesterol (TC). Of the 87 hypercholesterolemic persons 22 (25%) had positive AAB compared to 5 (6%) controls. Furthermore, 8 of the hypercholesterolemic patients had a serum TSH level above 5 mU/l, i.e. the had subclinical hypothyroidism, not diagnosed before, whereas thyroid function was normal in all normocholesterolemic persons. The new and unexpected finding was that the hypercholesterolemic persons had on average a significantly higher serum TSH than the controls, and this was true even when persons with positive AAB were excluded. There was a significant correlation between TC and serum TSH. It is concluded that hypothyroidism may not be an all-or-none phenomenon, and that many hypercholesterolemic persons with thyroid tests within the conventional normal range may have a slight impairment of their thyroid function.

Identification of cellular proteins that can interact specifically with the T/E1A-binding region of the retinoblastoma gene product.

The SV40 T antigen (T)/adenovirus E1A-binding domain of the retinoblastoma gene product (pRB) has been fused to S. japonicum glutathione S-transferase, and the chimera, bound to insoluble glutathione, was used to search for cellular proteins that can interact specifically with pRB. At least seven such proteins were detected in extracts of multiple human tumor cell lines. These proteins failed to bind to a family of pRB fusion proteins that harbor inactivating mutations in the T/E1A-binding domain and to the wild-type fusion protein in the presence of a peptide replica of the pRB-binding domain of T. Therefore, the binding of one or more of these proteins may contribute to the growth-suppressing function of pRB.

Clinical, laboratory and immunologic effects of the treatment of endemic goiter with T4, T3 and KI.

We treated 204 patients with endemic nontoxic goiter with T4, T3 and KI, singly or in combination. Definitely nodular goiters were excluded, since the possibility of autonomy would be increased. Goiter size was evaluated before and 6 months after treatment clinically in a blind way, i.e. the observer (always the same) did not know either the pretreatment goiter size or the treatment the patient had received. At the same time various laboratory parameters were recorded. All the active treatments (but not placebo) resulted in a highly significant decrease in the gland size. The effectiveness decreased in the following order: 1) T3 50 micrograms/d (most effective), 2) (T4 50 micrograms/d + T3 12.5 micrograms) x 2, 3) T4 150 micrograms + iodide 150 micrograms/d, 4) T4 75 micrograms + T3 18.75 micrograms/d, 5) T4 200 micrograms/d, 6) T3 37.5 micrograms/d, 7) Iodide 300 micrograms/d, 8) T4 150 micrograms/d, 9) Iodide 150 micrograms/d (least effective) and 10) Placebo (not effective). The results show that T4 200 micrograms and T3 50 micrograms are roughly equipotent, and slightly more effective than 300 micrograms of Iodide. Taking into consideration the side effects (increase in pulse rate, shortening of the Achilles tendon reflex) did not change the order of effectiveness in an important way. The clinical outcome correlated in general with the suppression of the 131I uptake (r = 0.220, p = 0.03) and the TRH test (r = 0.248, p = 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Polyoma small and middle T antigens and SV40 small t antigen form stable complexes with protein phosphatase 2A.

We have purified the 36 and 63 kd cellular proteins known to associate with polyomavirus middle and small tumor (T) antigens and SV40 small t antigen. Microsequencing of the 36 kd protein indicated that it was probably identical to the catalytic subunit of protein phosphatase 2A (PP2A). Identity was confirmed by comigration on two-dimensional (2D) gels and by 2D analysis of complete chymotryptic digests. In addition, PP2A-like phosphatase activity was detected in immunoprecipitates of wild-type middle T. Immunoblotting experiments, comigration on 2D gels, and 2D analysis of limit chymotryptic digests demonstrated that the 63 kd protein, present in the middle T complex in approximately equimolar ratio to the 36 kd protein, is a known regulatory subunit of the PP2A holoenzyme. Finally, the 36 kd PP2A catalytic subunit can be immunoprecipitated by anti-pp60c-src antisera only from cells expressing wild-type middle T. These results suggest that complex formation between PP2A and T antigens may be important for T antigen-mediated transformation.