Proteolysis of the human DNA polymerase epsilon catalytic subunit by caspase-3 and calpain specifically during apoptosis

Abstract

Human DNA polymerase epsilon (pol epsilon) normally contains a 261-kDa catalytic subunit (p261), but from some sources it is isolated as a 140-kDa catalytic core of p261. This shortened form possesses normal or somewhat enhanced polymerase activity and its significance is unknown. We report here that caspase-3 and calpain can form p140 from p261 in vitro and in vivo and that during early stages of apoptosis induced in Jurkat cells by staurosporine or anti-Fas-activating antibody, p261 is cleaved into p140 by caspase-3. At later stages, activated calpain might also contribute to this conversion. The sites of cleavage by caspase-3 have been identified, and mutations at these 'DEAD boxes' resulted in cleavage-resistant enzyme. Cleavage at these sites separates the 'N-terminal catalytic core' from the 'C-terminal' regions described for p261. Cleavage does not occur during necrosis or following exposure to H(2)O(2) or methanesulfonic acid methyl ester. p140 is unlikely to be able to functionally replace p261 in vivo, since it does not bind to PCNA or the other pol epsilon subunits.

Figure 1

Pol ɛ p261 interacts with the small subunit of calpain in a yeast two-hybrid screen. (A) Regions of the small subunit of calpain (residues 153–268) that were found to interact with p261 residues 1108–2256 during the initial yeast two-hybrid screen. (B) Residues of p261 that contribute to the interaction with calpain II as determined by further yeast two-hybrid assays using deletion constructs.

Figure 2

In vitro cleavage of p261 with purified rabbit m-calpain demonstrates that pol ɛ p261 is a substrate of calpain. Positions of full-length p261 and its cleavage product, p140, are denoted by an arrow and an asterisk, respectively. Positions of protein molecular mass markers are indicated to the left in kDa.

Figure 3

In vivo cleavage of pol ɛ p261 by activated calpain in Jurkat cells. Jurkat cells were treated with different doses of the calcium ionophore A23187 for 1 h and cell lysates were prepared as described in Materials and Methods. After SDS–PAGE and transfer, the nitrocellulose membrane was probed with monoclonal antibodies against pol ɛ p261 (A), PARP (B) or calpain II (C). Full-length proteins are indicated by an arrow and cleavage products by an asterisk [p140 in (A)] or an arrowhead [activated calpain II in (C)].

Figure 4

Time courses of protein cleavages in apoptotic Jurkat cells. After staurosporine treatment, cell lysates and DNA were prepared as described in Materials and Methods. Protein samples were separated by SDS–PAGE and transferred onto a nitrocellulose membrane which was then probed with antibodies against pol ɛ p261 (A), PARP (B), pol ɛ p59 (C), caspase-3 (D) or calpain II (E). The membrane was stripped between each probing. Positions of molecular weight markers are indicated to the left of each panel in kDa. Full-length proteins are denoted by an arrow and cleavage products by an asterisk [p140 in (A)], or an arrowhead [cleaved PARP, (B); activated caspase-3, (D); activated calpain II, (E)]. (F) DNA fragmentation during apoptosis. Chromosomal DNA was prepared from apoptotic Jurkat cells, separated by 2% agarose gel electrophoresis and stained with ethidium bromide. DNA markers are to the left in base pairs (bp).

Figure 5

Cleavage of p261 during anti-Fas-activating antibody-induced apoptosis. Jurkat cells were pretreated with inhibitors for 30 min prior to 0.5 µg/ml anti-Fas-activating antibody treatment for 3.5 h. After SDS–PAGE and transfer, the membrane was probed with antibodies against pol ɛ (top panel) and PARP (bottom panel). Full-length proteins are denoted by an arrow and cleavage products by an asterisk (p140, top panel), or an arrowhead (cleaved PARP, bottom panel).

Figure 6

No p140 was detected during necrosis or following cellular DNA damage. (A) pol ɛ p261 cleavage is not detected during necrosis. Jurkat cells were induced to undergo apoptosis or necrosis as described in Materials and Methods. Immunoblots show that the cleavage of p261 [(top panel, (A)] occurs only in apoptotic cells as manifested by the PARP cleavage [(bottom panel, (A)]. For cellular DNA damage, IMR-90 cells were exposed to H₂O₂ for 1 h (B) or MMS for 4 h (C). Immunoblots were probed for p140 generation [(top panels of (B) and (C)] or PARP degradation (bottom panels). Full-length proteins are indicated by an arrow and clevage products in (A) are shown by an asterisk (p140) or an arrowhead (PARP).

Figure 7

No p140 was detected throughout the cell cycle initiating from G₀ or early S phase. IMR-90 cells were synchronized and released from G₀ (A) or early S phase (B), extracts were prepared and immunoblots using monoclonal antibody 3C5.1 were performed as described in Materials and Methods. Full-length p261 is indicated by an arrow.

Figure 8

Caspase-3 cleaves pol ɛ p261 in vitro. Bacterial extracts (20 µl) containing recombinant caspase-3, caspase-1 or extracts from cells harboring the parental vector were incubated with 20 µl of partially purified HeLa pol ɛ for 2 h. Western blot analysis indicates three cleavage products of 240 and 90 kDa (noted by arrowheads) and a major product of 140 kDa (asterisk). Full-length p261 is marked with an arrow.

Figure 9

Determination of caspase-3 cleavage sites. Both wild-type and D→A mutant polypeptides corresponding to the full-length p261 residues 830–1605 (A) and 1–975 (B) were synthesized in vitro and then reacted with purified caspase-3. Reaction mixtures were separated by SDS–PAGE, transferred onto a nitrocellulose membrane and visualized by autoradiography. Full-length polypeptides are denoted by an arrow and cleavage products that are not present in the mutant are by arrowheads. (C) The two caspase-3 cleavage sites are located at the junctions between the three proposed domains (1).