We have shown that the budding yeast APC subunits, Cdc16, Cdc23, and Cdc27, are phosphorylated in vivo and in vitro. Phosphorylation in vivo depends on Cdc28, and in vitro it is catalyzed by pure Cdc28/Clb2/Cks1 complexes. Mutating potential Cdc28 phosphorylation sites in Cdc16, Cdc23, and Cdc27 abolishes their in vivo phosphorylation and compromises the mitotic, but not the G1 functions of the APC. We have also shown that Cdc5 affects APC phosphorylation in vivo and can catalyze APC phosphorylation in vitro. Our analysis of APC phosphorylation site mutants in vivo and in vitro, however, argues that in vivo Cdc5 indirectly induces the phosphorylation of Cdc16, Cdc23, or Cdc27, rather than directly modifying these subunits.
The APC Is Phosphorylated in Budding Yeast
Our results agree with studies on other organisms that show mitosis-specific APC phosphorylation. Cdc16, Cdc23, Cdc27, and Apc1 are phosphorylated in frogs; Apc1, Cdc16, and Cdc27 are phosphorylated in mammalian tissue culture cells; and Cdc16 (Cut 9) is phosphorylated in fission yeast (Peters et al. 1996
; Yamada et al. 1997
; Patra and Dunphy 1998
; Kotani et al. 1999
). Although APC phosphorylation has been shown to activate the Cdc20-dependent APC in mammalian tissue culture and clam egg extracts (Kotani et al. 1998
; Shteinberg et al. 1999
), and Cks1 depletions prevent mitotic exit in frog extracts (Patra and Dunphy 1996
), this is the first report to examine the in vivo function of APC phosphorylation. Although phosphorylation of Cdc16, Cdc23, and Cdc27 is not essential for viability in budding yeast, our studies suggest that it stimulates Cdc20-dependent APC activity and Cdc20 binding to the APC in vivo.
Cdc27 remains partially phosphorylated in G1 cells ( C). The presence of slower migrating Cdc27 in G1 cells could arise two ways: during the exit from mitosis, if Cdc28-catalyzed phosphorylation declines after phosphatases have been inactivated; or in G1, by phosphorylation catalyzed by another kinase. Because Cdc27-5A runs as a single band on Western blots in G1 (), we favor the possibility that G1 phosphorylation on Cdc27 remains from the previous mitosis. This finding would suggest that the phosphatase that removes phosphorylation from the APC is only active in mitosis. PP2A has been proposed to play such a role in clams and frogs (Lahav-Baratz et al. 1995
; Vorlaufer and Peters 1998
In one report, Plk has been identified as the major kinase of the mammalian APC (Kotani et al. 1998
), although others have argued that this role is played by Cdc2 (Lahav-Baratz et al. 1995
; Patra and Dunphy 1998
; Shteinberg and Hershko 1999
; Shteinberg et al. 1999
). We asked if its budding yeast homologue, Cdc5, plays a similar role. In vivo, APC phosphorylation is reduced in the cdc5-1
mutant and purified Cdc5 phosphorylates the APC in vitro ( C and 8 A). Three observations argue that in living cells Cdc5 does not directly phosphorylate the APC subunits we have studied: phosphorylation site mutations that completely block phosphorylation of Cdc16, Cdc23, and Cdc27 in vivo, do not block in vitro phosphorylation of these subunits by Cdc5 ( B); purified Cdc28/Clb2/Cks1, that lacks detectable Cdc5, efficiently phosphorylates immunoprecipitated APC (); and the same mutations that block Cdc28-catalyzed phosphorylation in vitro also block in vivo APC phosphorylation ().
If Cdc5 does not phosphorylate Cdc16, Cdc23, and Cdc27 directly, how does it regulate the phosphorylation of these subunits? Cdc5 may be responsible for phosphorylating other APC subunits (Apc1, -2, -4, and -5 are potential substrates; ) in vivo, and the phosphorylation of these subunits may affect the phosphorylation of the Cdc28 targets Cdc16, Cdc23, and Cdc27. Alternatively, Cdc5 may modulate Cdc28/Clb/Cks1 activity or localization.
Phosphorylation Stimulates Cdc20-dependent APC Activity
We have shown that phosphorylation site mutants in the APC reduce activation of the Cdc20-dependent APC. The half-life of Pds1 is increased in CDC27-5A
cells, and this defect in Cdc20 function could be explained by the observed inability of Cdc20 to bind an APC containing Cdc16-6A. This data supports genetic experiments showing that reduced mitotic Cdc28 activity compromises the Cdc20-dependent APC (Rudner et al. 2000
If Cdc20 binding and activity depend on a phosphorylated APC, why is the triple mutant CDC16-6A CDC23-A CDC27-5A
viable? Even in the triple mutant there is some residual Cdc20 binding to the APC (data not shown), which is presumably sufficient to drive the metaphase to anaphase transition. The residual binding of Cdc20 to the APC could depend on the phosphorylation of the other subunits. In support of this idea, we see weak phosphorylation of proteins we believe to be Apc1, -4, -5, and -9 in some in vivo labelings (data not shown), and a protein we believe to be Apc9 is phosphorylated in vitro by Cdc28/Clb2/Cks1 complexes (data not shown and ; Zachariae et al. 1996
). In addition, cdc28-1N
, a mutation in Cdc28 that cannot bind Cks1 (Kaiser et al. 1999
; and data not shown) and cks1-38
, have reduced APC phosphorylation ( C and 4 E). These two mutants are temperature-sensitive for growth and arrest in mitosis (Piggott et al. 1982
; Tang and Reed 1993
), suggesting that APC phosphorylation may be essential. Alternatively, it has been proposed that the primary defect in cdc28-1N
is in proteasome function (Kaiser et al. 1999
), though proteasome activity was examined in G1, not in mitosis, leaving the relevance of this finding to the exit from mitosis uncertain.
Our data suggests that activation of the APC by phosphorylation opposes its inhibition by the spindle checkpoint. Although CDC16-6A CDC23-A CDC27-5A is viable, its delay in mitosis ( A) becomes lethal when the spindle checkpoint is activated ( B). Both APC phosphorylation and the spindle checkpoint affect the ability of Cdc20 to activate the APC, but have no effects on the G1, Hct1-dependent activity of the APC.
Regulation of APC Phosphorylation
Phosphorylation of the APC in frogs and clams in vitro depends on homologues of the small Cdk binding protein, Cks1, and in clams, Cks1 stimulates Cdc20-dependent APC activity (Patra and Dunphy 1998
; Shteinberg and Hershko 1999
; Shteinberg et al. 1999
). In budding yeast, the role of Cks1 remains uncertain. Although we add purified Cks1 to our in vitro kinase reactions, Cks1 is not required for APC phosphorylation in vitro ( and data not shown). However, APC phosphorylation in vivo clearly depends on Cks1 ( E) and phosphorylation of Cdc27 is required for APC binding to Cks1-coupled beads ( D). We do not think that the binding of the APC to Cks1-coupled beads correlates with the ability of Cdc28 to phosphorylate the APC in vivo: although an APC containing Cdc27-5A does not bind to Cks1-coupled beads, Cdc16 and Cdc23 are fully phosphorylated in a CDC27-5A
mutant. Despite this in vivo finding, we do see reduced in vitro phosphorylation of Cdc16 and Cdc23 in an APC containing Cdc27-5A ( C).
Mutants that affect the mitotic form of Cdc28 have reduced levels of phosphorylation of the APC, whereas cdc28-4
cells, which are defective in the G1 form of Cdc28 (Reed 1980
), show normal phosphorylation of Cdc27 and Cdc16. We were surprised to discover that APC phosphorylation in cdc28-4
appears to be normal ( E), because this mutant has ~20% the amount of Cdc28 protein as wild-type cells at the permissive temperature of 23°C, and very little detectable Cdc28-associated kinase activity when immunoprecipitated from cell lysates (Surana et al. 1991
; and data not shown). A possible explanation of the absence of mitotic defects in cdc28-4
cells is that the specific activity of each Cdc28-4 molecule is equal to that of wild-type Cdc28, although the total number of active kinases is drastically reduced. The specific activity of individual Cdc28 molecules may be critical for APC phosphorylation because one Cdc28/Clb2/Cks1 complex may bind persistently to the APC. Once bound to the APC, this single complex might be responsible for multiple phosphorylations. If the steady state phosphorylation of the APC is determined by the balance between phosphorylation by Cdc28 and dephosphorylation by protein phosphatases, and Cdc28 remains bound to the APC, a drop in specific activity of Cdc28 would reduce the phosphorylation and activity of the APC.
How do cells escape from mitosis? If activating Cdc28/Clb/Cks1 complexes activates the Cdc20-dependent APC, which in turn triggers chromosome segregation, how do cells ensure that the lag between activating Cdc28 and activating the Cdc20-dependent APC is long enough to assemble a spindle and align chromosomes on it? Although one answer is that the spindle checkpoint inhibits Cdc20 in cells with misaligned chromosomes (Hwang et al. 1998
; Kim et al. 1998
), this explanation is not enough. Inactivating the spindle checkpoint does not kill yeast cells, implying other mechanisms exist to block premature activation of the Cdc20-dependent APC. Another possible mechanism is regulating the abundance of Cdc20. High levels of CDC20
transcripts are restricted to mitotic cells and APC-dependent proteolysis restricts the abundance of Cdc20 (Weinstein 1997
; Kramer et al. 1998
; Prinz et al. 1998
; Shirayama et al. 1998
). None of these forms of regulation exist in early frog embryos, where Cdc20 (Fizzy) levels are constant through the cell cycle and spindle depolymerization does not induce mitotic arrest (Minshull et al. 1994
; Lorca et al. 1998
). In addition, overexpressing Cdc20 in budding yeast raises the level of Cdc20 mRNA and protein, but does not advance the exit from mitosis, suggesting that other mechanisms must exist to regulate Cdc20-dependent APC activity (Prinz et al. 1998
). Regulating the rate of Cdc28-catalyzed APC phosphorylation provides an additional mechanism. If this phosphorylation were slow relative to spindle assembly, most cells would manage to align their chromosomes on the spindle before activating the Cdc20-dependent APC, which in turn induces Pds1 destruction and anaphase.
Note Added in Proof. Similar results showing that Cdc20 only binds to a phosphorylated APC have been published recently (Kramer, E.R., N. Scheuringer, V. Podrelejnikov, M. Mann, and J.M. Peters. 2000. Mol. Biol. Cell. 11:1555–1569).