The cyclin-dependent kinase inhibitor p27(Kip1) induces N-terminal proteolytic cleavage of cyclin A

Abstract

Progression through the cell cycle is regulated in part by the sequential activation and inactivation of cyclin-dependent kinases (CDKs). Many signals arrest the cell cycle through inhibition of CDKs by CDK inhibitors (CKIs). p27(Kip1) (p27) was first identified as a CKI that binds and inhibits cyclin A/CDK2 and cyclin E/CDK2 complexes in G1. Here we report that p27 has an additional property, the ability to induce a proteolytic activity that cleaves cyclin A, yielding a truncated cyclin A lacking the mitotic destruction box. Other CKIs (p15(Ink4b), p16(Ink4a), p21(Cip1), and p57(Kip2)) do not induce cleavage of cyclin A; other cyclins (cyclin B, D1, and E) are not cleaved by the p27-induced protease activity. The C-terminal half of p27, which is dispensable for its kinase inhibitory activity, is required to induce cleavage. Mechanistically, p27 does not appear to cause cleavage through direct interaction with cyclin/CDK complexes. Instead, it activates a latent protease that, once activated, does not require the continuing presence of p27. Mutation of cyclin A at R70 or R71, residues at or very close to the cleavage site, blocks cleavage. Noncleavable mutants are still recognized by the anaphase-promoting complex/cyclosome pathway responsible for ubiquitin-dependent proteolysis of mitotic cyclins, indicating that the p27-induced cleavage of cyclin A is part of a separate pathway. We refer to this protease as Tsap (pTwenty-seven- activated protease).

Figure 1

p27 induces formation of a modified form of cyclin A (cycA*). (A) p27 expression in RL leads to modification of cyclin A but not other human cyclins. Human p27 was transcribed and translated in rabbit RL (RL-p27) and incubated with HeLa-HU extracts (for detection of cyclins A, D1, and E) or HeLa-Noc extracts (for detection of cyclin B). HeLa-HU extract was incubated with unprogrammed RL as a control. Samples were taken at the indicated times and analyzed by SDS/PAGE followed by immunoblotting with antibodies against human cyclins A, D1, E, and B. (B) The ability to induce formation of cycA* is specific to p27. HeLa-HU extracts were incubated with RL in which the indicated human (RL-p15^Ink4b, RL-p16^Ink4a, RL-p21^Cip1, or RL-p57^Kip2) or Xenopus (RL-p28^Kix1) CKIs had been translated. Samples were taken at the indicated times and analyzed by SDS/PAGE followed by blotting with cyclin A antibodies.

Figure 2

Proteolytic cleavage yields an N-terminally truncated form of cyclin A lacking the mitotic destruction box (D box). (A) Diagram of cyclin A. The location of the D box and cyclin box are shown; the predicted site of p27-induced cleavage is indicated by asterisks. (B) p27 induces removal of N-terminal sequences from cyclin A. In vitro translated cyclin A protein bearing an N-terminal AU1 and a C-terminal HA tag was incubated with HeLa-HU extracts and RL-p27 to induce cleavage. Samples were taken at 0 and 120 min and were analyzed by SDS/PAGE followed by immunoblotting using antibodies against the AU1 or HA tag, as indicated. (C) Cyclin A is cleaved between amino acid residues 67 and 77. Radiolabeled cyclin A translation product was incubated for 240 min with RL-p27 to induce cleavage, as in B. A set of N-terminal deletion mutants of cyclin A (Δ1–57, Δ1–67, Δ1–77) was generated by PCR and translated in vitro to yield a set of radiolabeled cyclin A size markers. Samples were analyzed by SDS/PAGE followed by autoradiography. (D) R70 and R71 are essential for cleavage of cyclin A. Radiolabeled translation products of the indicated point mutants were mixed with HeLa-HU extracts and RL-p27 and incubated for 240 min. Samples were analyzed as in C. (E) Complete set of cyclin A point mutants tested for p27-induced cleavage. Mutants were tested as in D.

Figure 3

p27 induction of the cyclin A-cleaving activity. (A) p27 activates a protease that, once activated, no longer requires the presence of p27. p27 was translated in RL (RL-p27) and p27 was subsequently removed by immunodepletion. Immuno- or mock-depleted RL-p27 was incubated with HeLa-HU extracts for the indicated times, and generation of cycA* was monitored by immunoblotting. As controls, HeLa-HU extracts were incubated with nondepleted RL-p27 or nondepleted or immunodepleted unprogrammed RL as indicated (Upper). The efficiency of p27 immunodepletions from RL and from RL-p27 was examined by immunoblotting with p27 antibodies (Lower). (B) The N-terminal domain of p27, which contains its kinase inhibitory activity, is required but not sufficient to induce cleavage of cyclin A. Various deletion or point mutants of p27 (shown schematically on the right) were translated in reticulocyte lysate and incubated with HeLa-HU extracts. Samples were analyzed by SDS/PAGE followed by immunoblotting using cyclin A antibodies.

Figure 4

p27-induced cleavage of cyclin A and D-box-dependent degradation of cyclin A occur independently. (A) Cleavage of cyclin A is not a prerequisite for its degradation in human G₁-phase extracts. Radiolabeled wild-type cyclin A, D-box mutant (R47L50 → AA), or noncleavable mutant (T69R70 → AA) were produced by in vitro translation and incubated with HeLa G₁ cell extracts for the indicated times. Degradation of translation products was monitored by SDS/PAGE followed by autoradiography. (B) p27-induced cleavage of cyclin A is not dependent on a functional destruction box. Radiolabeled, in vitro translated wild-type cyclin A or D-box mutant proteins were incubated with HeLa-HU extract and RL-p27. Samples were analyzed as in A.

Figure 5

Cleaved cyclin A is stable and remains part of an active CDK complex. (A) Cleaved cyclin A is stabilized in G₁ cell extracts. Radiolabeled, in vitro translated full-length (RL-cycA) and a truncated cyclin A protein corresponding to cycA* (RL-cycA Δ1–70) were prepared and incubated with HeLa G₁ cell extracts as in Fig. 4A. Degradation of translation products was monitored by SDS/PAGE followed by autoradiography. (B) Cleaved cyclin A remains part of an active CDK complex. HeLa-HU extracts were coincubated with RL-p27 that had been immunodepleted of p27 for the indicated times. Cleavage of endogenous cyclin A was monitored by immunoblotting (Upper). At each indicated time point samples were taken and cyclin A-containing CDK complexes were recovered by immunoprecipitation; subsequent kinase assays used histone H1 as a substrate (Lower).

Figure 6

In vivo cleavage of cyclin A. (A) Cyclin A is cleaved in vivo in 293 (human) cells. 293 cells were transfected with empty vector (lane 1), wild-type cyclin A cDNA (lane 2), or a noncleavable mutant of cyclin A cDNA (lane 3). Extracts of transfected cells were analyzed by SDS/PAGE followed by immunoblotting with cyclin A antibodies. (B) Bromodeoxyuridine (BrdU) incorporation of FR3T3 (rat) cells after release from serum starvation. FR3T3 cells were serum starved for 72 hr and released into medium containing 10% serum. At each time point cells were pulse-labeled with bromodeoxyuridine to monitor DNA synthesis. (C) Detection of cycA* in FR3T3 cells. After release from serum starvation FR3T3 cells were taken at 2-hr intervals and processed for immunoblotting using mouse-specific cyclin A antibodies.