Tyrosine phosphorylation of p97. (A) LDM were incubated or not using tER assembly conditions as previously described (10–12) in the presence or absence of 100 μM bpV(phen). Solubilized membranes were immunoprecipitated either with antiphosphotyrosine antibodies (Ip: αPY, top gel) or with anti-p97 antibodies (Ip: αp97, middle and lower gels) followed by immunoblotting using anti-p97 (Ib: αp97, top and bottom gels) or anti-phosphotyrosine antibodies (Ib: αPY, middle gel) and revealed by chemiluminescence (ECL). A representative experiment is shown (n = 4) with similar results observed each time. (B) LDM were incubated in the same medium described above but containing 100 μCi [γ-³²P]ATP and the absence or presence of either 100 μM bpV(phen) or 200 μM genistein. After p97 immunoprecipitation, the proteins in the immunoprecipitate were separated by SDS/PAGE and transferred onto nitrocellulose membrane. The membrane was subjected to radioautographic analysis using X-Omat AR film (top gel), then probed with anti-p97 and revealed by ECL (bottom gel). Densitometric analysis of the x-ray films was performed, and the representation is the mean of three independent experiments ± SD for the three incubation conditions.

Purification of tyrosine kinase activities from LDM. (A) Low-density microsomes from rat liver (600 μg to 5 mg) solubilized were incubated with ATP-agarose beads. Eluted fractions were then tested for the presence of kinase activity by incubation with 0.5 mg/ml poly Glu-Tyr, and radioactivity was measured by scintillation counting. Protein concentration was measured for each fraction and is reported on the graph. L represents 5% of total protein lysate chromatographed on ATP-agarose. Fractions containing the highest specific activity were concentrated and either in vitro autophosphorylated (Right, lane 1) or directly resolved by SDS/PAGE followed by immunoblotting with anti-JAK-2 antibodies (Right, lane 2). (B) In vitro phosphorylation of JAK-2 immunoprecipitate from LDM (lane 1, overnight exposure) or in vitro phosphorylation of JAK-2 immunoprecipitate from LDM, followed by release and sequential immunoprecipitation with anti-JAK-2 antibodies (lane 2, overnight exposure) or anti-p97 antibodies (lane 3, 40-h exposure). In lane 4, a nonimmune serum (NI) was used in the primary immunoprecipitation, and an anti-p97 was used for the secondary immunoprecipitation (overnight exposure). (C) In vitro phosphorylation of 10 μg of myelin basic protein (MBP, lane 1) or purified p97 (lane 2) by JAK-2 immunocomplex from LDM. A representative experiment is shown (n = 2).

Identification of p97 tyrosine phosphatase from low-density microsomes. (A) Cytosol or LDM were incubated as described above in the presence or in the absence of 100 μM bpV(phen) and 100 μCi [γ-³²P]ATP. Lysates were then incubated in the presence of glutathione-Sepharose beads carrying 15 μg of GST-PTPH1 D811A mutant for 4 h at 4°C. Proteins pulled-down were resolved by SDS/PAGE and either immunoblotted with anti-p97 or anti-GST antibodies and revealed by enhanced chemiluminescence (Middle and Bottom) or directly radioautographed to X-Omat AR films (Top). A representative experiment is shown (n = 3). (B) Analytical fractionation by isopycnic density gradient centrifugation of LDM. Each fraction was tested for its content p97, PTPH1, JAK-2, and Bip by immunoblotting. Quantitation was done as described under Experimental Procedures.

Effect of bpV(phen) and genistein on transitional ER assembly. LDM (A) were incubated using assembly conditions as previously described (B) in the presence of 100 μM bpV(phen) (C) or 350 μM genistein (D) for 180 min at 37°C. After the incubation, membranes were fixed and processed for electron microscopy. rm, rough membrane; st, smooth tubules. Scale bars represent 0.5 μm. (E) Morphometric studies of the amount of rough endoplasmic reticulum and smooth endoplasmic reticulum membranes in tER networks reconstituted in presence or absence of 100 μM bpV(phen) or 350 μM genistein. Average values (±SD) are shown from three separate experiments. RER, rough endoplasmic reticulum; SER, smooth endoplasmic reticulum. (F) Amount of tER assembly per total membrane reconstituted in presence or absence of 100 μM bpV(phen) or 350 μM genistein. Average values (±SD) are shown from three separate experiments. The mean number of networks was not significantly different when compared between control and DMSO (P > 0.05, n = 21) but was significantly different when compared between control and bpV(phen) (P < 0.005, n = 27) or DMSO and genistein (P < 0.001, n = 20). The amount of tER networks counted was as follows: control, 112 networks on 13 micrographs; bpV(phen), 50 networks on 14 micrographs; DMSO, 90 networks on 8 micrographs; and genistein, 219 networks on 12 micrographs. Results were expressed as average number of tER networks per 200 μm² of membrane pellicules.

(A) Association of p97 to the membranes is regulated by its tyrosine phosphorylation. LDM were incubated as previously described (10–12) in the presence or absence of 100 μM bpV(phen) or 350 μM genistein for different periods. The samples were then placed on ice and received 1 ml each of 0.25 M sucrose containing 3 mM imidazole. The samples were then centrifuged at 100,000 × g for 30 min. The proteins in the membrane pellets were dissolved directly in Laemmli buffer. The proteins in the supernatant fractions were concentrated by trichloroacetic acid precipitation. Proteins were then separated by SDS/PAGE, followed by immunoblotting with anti-p97 antibodies. (B) Densitometric analysis of the above immunoblots. Each value is the mean of three independent experiments ± SD. Closed symbols stand for membrane-associated p97 and open symbols for p97 released from the membranes, respectively, ♦,⋄ CTL; ▴,▵, genistein; ■,□, bpV(phen). (C) LDM (600 μg) were first incubated 45 min on ice as previously described, except that Hepes was replaced by 6 mM imidazole, pH 7.4; this incubation buffer contained or not 350 μM genistein or 30 μg of GST-PTPH1 or GST-PTPH1D811A or 100 μM bpV(phen). Microsomes were then solubilized in the same buffer containing 1.5% CHAPS. After spinning in a microfuge for 15 min at maximal speed, lysates were immunoprecipitated with anti-p97 antibodies. Immunoprecipitates were then resolved by SDS/PAGE, transferred onto nitrocellulose, and immunoblotted with either anti-syntaxin 5 antibodies or anti-p97 antibodies and revealed by ECL. A representative experiment is shown (control, genistein, bpV(phen), and GST-PTPH1DA, n = 3; GST-PTPH1, n = 2). Quantitation was made by scanning densitometry of the x-ray films and represents the mean ± SD (when n = 3); * indicates significant values P < 0.04. For syntaxin 5, the intensities corresponding to the three bands visualized were added. (D) Representative scheme of the sequence of events regulating p97 association with LDM.