Cleavage of cyclin A at R70/R71 by the bacterial protease OmpT

Abstract

Previous work has shown that cyclin A can be cleaved at Arg-70/Arg-71 by a proteolytic activity present in an in vitro-coupled transcription/translation system by using rabbit reticulocyte lysate programmed by plasmid DNA encoding p27(KIP1), a cyclin-dependent kinase inhibitor, but not by plasmid DNAs encoding other cyclin-dependent kinases inhibitors. Here we report that cyclin A is also cleaved by translation product programmed by plasmid DNA encoding cyclin B. Several findings indicate that the cleavage activity in this assay is provided by the bacterial protease OmpT, which cofractionates with cyclin B and p27(KIP1) plasmid DNAs and is thus carried over into the coupled in vitro transcription/translation reactions. (i) Cleavage activity appeared even when transcription or translation of the cyclin B or p27(KIP1) was blocked. (ii) Activity resembling OmpT, a serine protease that cleaves between dibasic residues, routinely copurifies with p27(KIP1) and cyclin B plasmid DNAs. (iii) Both cyclin A cleavage activity and OmpT activity are heat stable, resistant to denaturation, and inhibited by Zn(2+), Cu(2+), or benzamidine. (iv) Cyclin A cleavage activity is detected when using lysates or DNAs prepared from Escherichia coli strains that contained OmpT but not with strains lacking OmpT. (v) Purified OmpT enzyme itself cleaves cyclin A at R70/R71. These data indicate that OmpT can be present in certain DNA preparations obtained by using standard plasmid purification protocols, and its presence can potentially affect the outcome and interpretation of studies carried out using in vitro-translated proteins.

Figure 1

Cleavage of cyclin A by cyclin B translated in rabbit RL. (A) Cyclin B(RL) induces efficient cleavage of cyclin A(CΔ114). GST-cyclin A(CΔ114) was mixed with unprogrammed RL (lanes 2 and 3) or cyclin B(RL) (lane 4). The reactions were incubated at 37°C and stopped with SDS-sample buffer at the indicated time. The proteins were analyzed by SDS/PAGE and Coomassie blue staining. Molecular size standards (lane 1) in kDa are indicated on the left. The positions of GST-cyclin A(CΔ114) and the cleaved form of cyclin A (*) are indicated on the right. (B) Cleavage of cyclin A expressed in mammalian cells into two fragments by cyclin B(RL). FLAG-cyclin A was transiently transfected into HtTA1 cells. Cell extracts were prepared and the expressed cyclin A were immunoprecipitated with anti-FLAG antibodies. The immunoprecipitates were incubated with unprogrammed RL or cyclin B(RL) as indicated. The samples were applied onto 12.5% SDS/PAGE (top two panels) or Tricine gel (bottom two panels), and subjected to immunoblotting with anti-FLAG mAb M2, anti-cyclin A monoclonal E23, or M2 and E23 together as indicated. In this paper, the N-terminal fragment of cyclin A is denoted with “*”, and the C-terminal fragment is denoted with “**” (see main text).

Figure 2

Cyclin A cleavage is abolished by R70A + R71A mutation. (A) RL expressing cyclin B, cyclin A, or cyclin A(R70A + R71A) were mixed as indicated. LLnL (50 μM) or protease inhibitor (PI) mixture was included in the reactions where indicated. The reactions were incubated for the indicated time before stopped with SDS sample buffer. The samples were analyzed by SDS/PAGE and phosphorimagery. The positions of the molecular size standards (in kDa), the expressed proteins, and the cleaved form of cyclin A (**) are indicated. (B) FLAG-cyclin A (lanes 1–3) or R70A + R71A mutant (lanes 4–6) were transiently transfected into HtTA1 cells. Cell extracts were prepared, and the expressed cyclin A were immunoprecipitated with anti-FLAG antibodies. The immunoprecipitates were incubated with unprogrammed RL or cyclin B(RL) as indicated. Cleavage of cyclin A was analyzed by immunoblotting with anti-cyclin A mAb E23 (Upper), and expression of cyclin B was analyzed with phosphorimagery (Lower).

Figure 3

RL-translated cyclin B protein is not required to induce cyclin A cleavage. (A) Blocking cyclin B synthesis does not affect cyclin A cleavage. Cyclin B DNA was mixed with RL either in the absence or presence of cycloheximide (1 mg/ml). No cyclin B translation was detected when cycloheximide was added (Lower). The RL were then incubated with GST-cyclin A(CΔ114) for the indicated time, and cleavage of cyclin A was detected by SDS/PAGE and Coomassie blue staining (Upper). (B) Cyclin A proteolytic activity is present in the DNA and DN5α lysate. GST-cyclin A(CΔ114) was incubated with buffer, cyclin B(RL), cyclin B DNA, DH5α lysate, or BL21(DE3) lysate as indicated. GST-cyclin A(CΔ114) was not added in the reactions in lanes 6 and 8. Cleavage of cyclin A was detected by SDS/PAGE and Coomassie blue staining. Molecular size standards (lane 1) in kDa are indicated on the left.

Figure 4

RL-translated p27^KIP1 is not required to induce cyclin A cleavage. (A) Cyclin A translation product was produced by the addition of cyclin A plasmid DNA to the RL-coupled transcription/translation system. p27^KIP1 plasmid DNA was added to coupled transcription/translation RL in the absence or presence of cycloheximide and RNA polymerase as indicated. Reactions were mixed and incubated as indicated and analyzed by SDS/PAGE followed by autoradiography. (B) p27^KIP1 plasmid DNA was prepared from E. coli strain DH5α (lanes 1 and 2) and BL21 (lanes 3 and 4). The plasmid DNA preps were mixed with cyclin A(RL) for the indicated time.

Figure 5

Cyclin A proteolytic activity is present in DNA preparation and in bacterial lysates. (A) Inhibition of cleavage of cyclin A by benzamidine. GST-cyclin A(CΔ114) was incubated with cyclin B(RL) in the presence of buffer (lane 2), E64 (lane 3), benzamidine (lane 4), or soybean trypsin inhibitor (lane 5) at 37°C for 120 min. Cyclin A cleavage was detected by SDS/PAGE and Coomassie blue staining. Molecular size standards in kDa are indicated on the left. (B) Cyclin B in pET21d DNA (lanes 4–9) were boiled for 5 min (lane 5), subjected to ethanol precipitation (lane 8), or treated with benzimidine (lane 6), DNase (lane 7), or proteinase K (lane 9). The proteinase K was subsequently inactivated by phenol/chloroform extraction. The samples were then incubated with purified GST-cyclin A(CΔ114) for the indicated time. Cleavage of cyclin A was detected by SDS/PAGE and Coomassie blue staining (Upper). Molecular size standards (lane 1) in kDa are indicated on the left. DNA in the samples was visualized by agarose gel electrophoresis and ethidium bromide staining (Lower). (C) GST-cyclin A(CΔ114) was incubated with buffer (lanes 2 and 3), or DH5α lysates (lanes 4–9) in the presence of ZnCl₂ (0.1 mM, lane 5; 1 mM, lane 6) or CuCl₂ (0.1 mM, lane 7; 1 mM, lane 8; 10 mM, lane 9). Cleavage of cyclin A was detected by SDS/PAGE and Coomassie blue staining. Molecular size standards (lane 1) in kDa are indicated on the left.

Figure 6

Cleavage of cyclin A by OmpT. (A) GST-cyclin A(CΔ114) was incubated with buffer (lanes 2 and 3), lysates of BL21(DE3) (lane 4), BL21(DE3) transformed with cyclin B construct (lane 5), or transformed with OmpT expression construct (lane 6) for the indicated time. Cleavage of cyclin A was detected by SDS/PAGE and Coomassie blue staining. Molecular size standards (lane 1) in kDa are indicated on the left. (B) Purified OmpT protein (lanes 4–6) was incubated with GST-cyclin A(CΔ114) (lanes 2–5) for the indicated time. Cleavage of cyclin A was detected by SDS/PAGE and Coomassie blue staining. Molecular size standards (lane 1) in kDa are indicated on the left.