Identification of M2-PK as E7-binding protein. (A) Plasmid pLexA-16 E7(39–98)∷HIS3 (18) encodes the DNA binding domain of LexA fused in-frame to the C-terminal part of HPV-16 E7 and the HIS3 selectable marker. The structure of the expression library, derived from vector pJG4–5 (19) by insertion of cDNA fragments from a WI-38 cDNA library, is indicated. B42-TAD refers to the B42 transactivation domain. B42 fusion proteins are expressed from the inducible GAL1 promotor, and these plasmids contain the TRP1 gene as selectable marker. (B) The plasmid designated pB42-M2-PK∷TRP1 was isolated during the interaction screen; it contains the full-length human cDNA for M2-PK, as described by Tani et al. (43). (C) Derivatives of yeast strain EGY48/pSH1834, expressing various LexA fusion proteins as indicated, were transformed with the plasmid pB42-M2PK∷TRP1. pJG4–5, expressing the unfused B42 trans-activation domain, was used as negative control. Transformants were selected for uracil, histidine, and tryptophane prototrophy and grown in glucose minimal medium (Ura⁻, His⁻, Trp⁻). Yeast cells then were streaked out onto each of three plates and incubated for 4 days at 30°C under the following nutrient conditions (19). Control: glucose minimal medium with leucine; all strains grow. Glucose: glucose minimal medium without leucine; selection for B42 fusion protein independent activation of the LexAo6-LEU2 reporter. Galactose: galactose minimal medium without leucine, selecting for B42 fusion protein dependent activation of the LexAo6-LEU2 gene.

E7 domains required for M2-PK binding. (A) Structure of the E7 proteins. Conserved domains 1 and 2 (cd1 and cd2) and the putative zinc finger motifs (CXXC) of the E7 proteins of HPV-11 and HPV-16 (44) are indicated. (B) Saccharomyces cerevisiae strain EGY48/pSH1834, containing a LexA operator-lacZ gene (LexAo8-Gal1-LacZ∷URA3), was transformed with the plasmid pB42-M2PK∷TRP1. Plasmids encoding LexA fusion proteins were coexpressed, as indicated. β-galactosidase activity was measured in cellular extracts. LexA-Bicoid and LexA-C-Myc(C-term) were used as negative controls. Extracts from yeast strains expressing the B42-HA1 (C) or B42-HA1-M2PK (D) fusion proteins were incubated with purified GST or GST-E7 fusion proteins and immobilized on glutathione Sepharose 4B beads. Bound proteins were separated on a 12.5% SDS-polyacrylamide gel and analyzed by Western blotting using anti-HA1 antibodies. Lysate refers to 5 μg of yeast whole-cell extract that was loaded as input control.

Interaction of M2-PK and HPV-16 E7 in mammalian cells. (A-E) Extracts were prepared from E7/2 and M/1 cells and subjected to immunoprecipitation by mAbs to HPV-16 E7, human M2-PK, and GST (control). Total cell lysate (60 μg) was loaded as input control. (A and B) Immunoprecipitation with monoclonal anti-HPV-16 E7 antibodies; Western blot was performed with anti-M2-PK antibodies (A) and antibodies to HPV-16 E7 (B). (C) Immunoprecipitation with monoclonal control antibodies; Western blot was performed with monoclonal anti-M2-PK antibodies. (D and E) Immunoprecipitation with monoclonal anti-M2-PK antibodies; Western blot was performed with anti-M2-PK (D) and anti-HPV-16 E7 (E) antibodies. (F) Western blot analysis of the expression of the Δ79–83 mutant of HPV-16 E7 in NIH 3T3 subclone Δ79–83. IgGh, immunoglobulin G heavy chain; IgGl, immunoglobulin G light chain.

Modulation of M2-PK activity by HPV-16 E7. (A) Overexpression of M2-PK in 14/2 cells. Expression of E7 was induced in 14/2 cells by addition of dexamethasone for 4 hr. In a control experiment, asynchronously growing NRK cells were either mock-treated or treated with dexamethasone for 4 hr. Extracts were prepared from these cells as indicated, and the abundance of M2-PK in the cellular extracts was analyzed by direct immunoblotting. (B Upper) Gel filtration analysis. Extracts from E7-expressing or control 14/2 cells were subjected to gel filtration, followed by determination of M2-PK activity in individual fractions, by using a PEP concentration of 2 mM. The peaks of main activities also are indicated for GAPDH, enolase (EN), and lactate dehydrogenase (LDH), assayed in parallel. (Lower) The abundance of M2-PK in each fraction was determined by direct immunoblotting. (C and D) Isoelectric focusing. Extracts from E7-expressing or control 14/2 cells were subjected to isoelectric focusing, and M2-PK activity was determined in the presence of 2 mM PEP (C) or 0.2 mM PEP (D) as indicated. The peak of main activity also is indicated for GAPDH, assayed in parallel. pI values of selected fractions are given for reference.

Modulation of M2-PK by E7 in vitro. (Upper) Extracts from M/1 cells were incubated with GST (90 ng/μl) or GST-HPV16E7 (90 ng/μl) for 1 hr at 4°C and subjected to gel filtration. Activity of M2-PK was measured in all fractions in the presence of 2 mM PEP. On addition of GST-HPV16E7, the proportion of tetrameric M2PK was reduced from 63% to 54%, and the proportion of dimeric enzyme increased from 37% to 46%. For calibration of the column, the maximal activities of GAPDH, lactate dehydrogenase (LDH), and enolase (EN) were determined in the individual fractions. (Lower) The distribution of M2-PK and GST-16E7 in the fractions was monitored by direct immunoblotting using antibodies against M2-PK and GST, respectively.