- Email: [email protected]
Cdc2 regulatory factors
Cdc2 regulatory
factors
Thomas R Coleman and William G Dunphy California
Institute of Technology,
Pasadena,
USA
A growing family of kinases and phosphatases controls the activity of the cyclin-dependent kinase cdc2. The past year has seen the identification of the cdk activating kinase as well as considerable elucidation of the cdc25/weel regulatory pathways. Both cdc25 and wee1 appear to be regulated by upstream kinase/phosphatase networks. In addition, it is likely that other regulatory mechanisms cooperate with the weel/cdc25 phosphorylation systems to control the action of cdc2. Together, these elaborate checks and balances ensure that cdc2 triggers mitosis at the appropriate time. Current
Opinion
in Cell Biology
1994,
6:877-882
an enzyme known as cdk activating kinase (CAK). In contrast, phosphorylation on either Thrl4 or Tyr15 by multiple weel-like kinases dominantly inhibits cdc2 activity. At the onset of mitosis, the cdc25 phosphatase activates cdc2 by dephosphorylating Tyr15 and most probably Thrl4. Exit from mitosis occurs with degradation of the cyclin, dephosphorylation of Thrl61, and consequent inactivation of MPF (Fig. 1). This review focuses mainly on the regulation of cdc25 and weel.
Introduction In all eukaryotes, cyclin-dependent kinases (cdks) regulate each phase of the cell cycle (for background reviews, see [l-3]). The first discovered cdk, the cell division cycle 2 (cdc2) kinase, serves as the catalytic component of maturation promoting factor (MPF), a dominant regulatory factor that initiates mitosis by phosphorylating a wide variety of structural and regulatory proteins. Control of the cdc2 kinase is a highly regulated process which requires binding to a regulatory subunit (cyclin B) and concomitant phosphorylation at multiple, highly conserved residues. Phosphorylation on Thrl61, which is necessary for cdc2 kinase activation, is mediated by
CAK: the cdc2 activating
kinase
Activation of the cdc2-cyclin CAK-mediated phosphorylation
heterodimer requires of Thrl61. The crys-
Interphase Thr 161
Thr 14
Interphase
Tyr 15
rl
rl
cdc2
cdc2 cdc25
Dephosphorylation
,
3
k%Y m
I
Kinases
I
cyclin I
-----,
r----I I
I
cyclin I I I I I _ - - - - - - - - - _’ Degradation Active
MPF
8 1994 Current Opinion in Cell Biology
Fig. 1. Cdc2 and cyclin B control entry into mitosis. During interphase, newly synthesized cyclin B binds to cdc2 inducing its phosphorylation on three conserved residues. CAK activates cdc2 by phosphorylating the Thrl61 site; concomitantly, several Thrl4/TyrlS kinases dominantly inhibit cdc2 by phosphorylating the Thr14 and Tyrl5 sites. The cdc25 phosphatase induces mitosis by dephosphorylating these inhibitory phosphates, producing active MPF. Exil from mitosis occurs with degradation of cyclin and dephosphorylation of Thrl61 by an unknown phosphatase.
Abbreviations CAK-cdk activating MPF-maturation
kinase; CaMKlI-caIcium/calmoduIin promoting factor; MPM-2-mitosis
0 Current
kinase II; cdkprotein monoclonal
Biology
cyclin-dependent kinase; antibody-2; PPZA-protein
ltd ISSN 0955-0674
ME-MPM-2 phosphatase
epitope; 2A. 877
878
Cell multiplication
tal structure of human cdk2 reveals that the Thr160 residue (analogous to Thr161 of cdc2) resides on a structural loop that occludes the substrate-binding cleft. Thus, phosphorylation on this residue may alter the geometry of this inhibitory loop and stabilize an active conformation [4**]. Three groups [5*-7.1 determined that CAK is composed of a complex of at least one regulatory subunit (p37) and a catalytic subunit (M015). Interestingly, MO15 is structurally related to the cdc2 kinase, and the p37 regulatory subunit shares limited homology with the cyclin family. Together these two components are suffrcient to reconstitute CAK activity in a highly purified system [8**]. A proposal to rename MO15 as cdk7 and its p37 binding partner as cyclin H has been suggested [W]. The regulation of Thr161 phosphorylation has been a matter of widespread interest. Lee and co-workers [9] initially proposed that an inhibitor of MPF activity directly opposed the action of CAK by dephosphorylating Thr161. More recent data from this group, however, show that this inhibitor is a conventional form of protein phosphatase 2A (PP2A) that inhibits MPF by a less direct means. The addition of exogenous PP2A to Xenop~rs egg extracts inhibits Thrl61 phosphorylation in a dose-dependent manner during the lag period before mitosis, when the pivotal reaction(s) that trigger mitosis are expected to occur [lO**]. These findings suggest that PP2A may act indirectly on CAK or the Thrl61directed phosphatase (Fig. 2). This proposal, however, is apparently at odds with the fact that Thrl61 phosphorylation is constitutive throughout the cell cycle [ll]. Finally, the identity of the phosphatase that dephosphorylates Thr161 is not known, but a recently discovered phosphatase that can bind cdks (named Cdil, Cip2, or KAP [12-141) is a potential candidate.
Membrane-bound 161 14 15
al N
Active
Inactive
wee1
C
wee1
Inactive
wee1
0 1994 Current Opinmn in Cell Biology
Fig. 2. Multiple kinases and phosphatases prepare cdc2 and cyclin for activation. CAK stimulates cdc2 activity by phosphorylating (P) the Thr161 residue of cdc2. Both wee1 and a membrane-bound kinase phosphorylate the inhibitory residues Thrl4 and Tyrl5. Wee1 can be inactivated by several phosphorylation events. The niml kinase mediates one such event by phosphorylating the catalytic (carboxy-terminal, C) domain. An unknown phosphatase opposes niml to produce active weel. Multiple kinases tcdc2 and kinase X) most probably mediate the second inhibitory event by extensively phosphorylating the regulatory (amino-terminal, N) domain. This amino-terminal inhibitory phosphorylation is opposed by a PPZA-like activity.
ulated by some other pathway that operates in parallel with the accumulation of cyclin.
Regulation
of the wee1 protein
kinase
Genetic studies in yeast that demonstrated that the wee1 kinase is negatively regulated by the niml kinase [15,16] were validated by recent biochemical analyses. Specifically, the recombinant niml protein directly phosphorylates and inactivates isolated fission yeast wee1 in vitro [170-l ‘91. Interestingly, this niml-catalyzed phosphorylation of wee1 occurs in the carboxy-terminal region [17*], the area that contains the kinase domain. To date, niml homologues have not been identified in other species, and experimental evidence indicates that Xenopus egg extracts appear to lack a niml-like activity (T Coleman, W Dunphy, unpublished data). Therefore, a general role for niml in the control of wee1 activity remains to be demonstrated. Similarly, the question of what regulates yeast niml activity during the cell cycle remains unanswered. As wee1 is downregulated during mitosis when MPF is activated, MPF is one candidate for a niml activator. Alternatively, niml may be reg-
Whether or not niml homologues are present in other eukaryotes, phosphorylation by niml is not the sole mechanism through which wee1 is regulated. For example, deletion of the yeast nirrr1 gene is not lethal, but rather results in a prolonged Gz phase [15]. Direct evidence demonstrating the existence of a second wee1 regulatory pathway comes 13om a heterologous system in which fission yeast wee1 was introduced into Xerropus egg extracts [20*]. Mitotic, but not interphase, egg extracts contain a kinase which extensively phosphorylates the amino-terminal region of fission yeast weel. Like wee1 that has been phosphorylated in the carboxyterminal domain by niml, the.weel protein phosphorylated in its amino-terminal region is almost completely inactive as a cdc2-specific tyrosine kinase [20*]. An attractive candidate for the weel-inhibitory kinase is MPF Extensive phosphorylation of weel, however, can occur in mitotic extracts depleted of cdc2, suggesting that cdc2 is not the sole kinase responsible for inactivation of wee1 at mitosis [20-l. The phosphatase that serves to main-
Cdc2
regulatory
factors
Coleman
and Dunphy
tain wee1 in its active, underphosphorylated form during interphase possessesseveral hallmarks characteristic of a PP2A-like activity [10**,20*] (Fig. 2).
will therefore be necessary to elucidate the relationship between the replication checkpoint and cdc2 tyrosine phosphorylation.
The more recent characterization of a Xetqns wee1 kinase confirms the existence of a wee1 regulatory kinase/phosphatase pathway in an entirely homologous system (P Mueller, T Coleman, W Dunphy, unpublished data). The activity of endogenous Xerroprrswee1 protein is stringently regulated during the cell cycle: the interphase underphosphorylated form etliciently phosphorylates cdc2 exclusively on Tyrl5. whereas the mitotic hyperphosphorylated form is nearly inactive. Dephosphorylation of the mitotic form by treatment with PP2A restores its kinase activity. Moreover, in vitro treatment of Xcrrtipns wee1 with active cdc2-cyclin complex results in phosphorylation and inactivation ofweel kinase. At least one additional wee1 inhibitory kinase probably exists, however, as Xcnopprrswee1 becomes partially phosphorylated in mitotic extracts that have been depleted of active cdc2. Candidates for this weel-specific kinase include the MPM-2 epitope (ME) kinases that generate phosphopeptide epitopes on a large number of proteins recognized by the mitosis protein monoclonal antibody, MPM-2. The identities of the ME kinases have not been definitively established (21-231 but evidence suggests that ME kinase activity phosphorylates both fission yeast and Xenopns wee1 during mitosis (P Mueller, T Coleman, W Dunphy, unpublished data).
In vertebrates, cdc2 is also phosphorylated on Thrl4, the residue that adjoins Tyrl5, but the identity of the inhibitory kinase that phosphorylates cdc2 on this residue remains unclear. Interestingly, Kornbluth et al. [25*] reported a membrane-associated kinase activity that can phosphorylate Thr14 (a co-fractionating activity also phosphorylates Tyr15). It will be interesting to learn in which cellular membrane this kinase resides. Specific membrane localization may target the kinase to certain cdc2-cyclin subpopulations, allowing for differential activation/inactivation of MPF within the cell.
Another issue in wee1 regulation concerns the replication checkpoint. In Xmop~ts egg extracts, incomplete DNA replication blocks the activation of cdc2 by maintaining it in a tyrosine-phosphorylated form. It has been proposed that the control system that monitors DNA replication acts through a cdc2-specific tyrosine kinase [24]. In direct measurements, however, the activity of Xewpus wee1 does not appear to be modulated by the presence of unreplicated DNA (P Mueller, T Coleman, W Dunphy, unpublished data). Further studies
Inactive
Regulation
of
the cdc25 phosphatase
It is now well established that the cdc25 phosphatase is weakly active during interphase and fully active during mitosis [26,27]. Moreover, cdc25 activity is regulated by a kinase/phosphatase system: the active form has an extensively phosphorylated amino-terminal domain. As in the wee1 regulatory pathway, the cdc25 regulatory phosphatase appears to be a PP2A-like protein (Fig. 3). Furthermore, cdc25 can be activated following in vitro phosphorylation by active cdc2-cyclin complex [28,29]; the phosphorylation sites are consistent with those characterized in viva on mitotically active cdc25 [29,30-l. Interestingly, Xenopus extracts deficient in cyclin B can also phosphorylate cdc25 in the presence of the phosphatase inhibitor okadaic acid [27]: these results suggest the presence of additional cdc25 kinases. Likewise, Kuang and co-workers [31*] have shown that cdc25 is a major substrate of the ME kinase at mitosis. Thus a variety of kinases, including cdc2 and ME kinase(s), may contribute to cdc25 regulation, as is the case for the wee1 protein.
cdc25
Nuclear
envelope
Spindle
apparatus
Chromosomal
DNA
8 1994 Curmnl Gpinion in Cell Biology
Fig. 3. At the Gz/M transition, a positive feedback loop activates the cdc25 phosphatase and inactivates wee1 resulting in abrupt entry into mitosis. Inactive triplyphosphorylated cdc2-cyclin complexes can be activated by dephosphorylating the Thrl4 and Tyr15 residues, a process mediated by active cdc25 and opposed by the wee1 kinase. The cdc25 phosphatase oscillates between an amino-terminally phosphorylated (active) form and an underphosphorylated (inactive) form. Cdc25 activity is opposed by a PPZA-like activity and favored by an unknown kinase X fan ME kinase?) and MPF, which also drives this reaction forward by phosphorylating and inactivating the wee1 kinase. MPF promotes mitosis by acting on the nuclear envelope, the spindle apparatus and the chromosomal DNA.
879
;80
Cell multiplication
Many striking parallels exist between the regulation of wee1 and that of cdc25. Both proteins are’extensively phosphorylated during the G2/M transition. Phosphorylation of wee1 abolishes its kinase activity, while phosphorylation of cdc25 augments its phosphatase activity. As both proteins are phosphorylated in parallel, they may be controlled by the same kinase/phosphatase system. Indeed, both wee1 and cdc25 are dephosphorylated by a PP2A-like activity, and the cdc25-specific PP2Alike enzyme appears to be less active during mitosis [27]. Similarly, both proteins are substrates for the kinase activity that they control (MPF), thus suggesting an appealing positive-feedback loop wherein a small amount of active cdc2 would rapidly and irreversibly trigger mitosis (Fig. 3). Because the activities of cdc25 and wee1 are quite probably regulated by their ultimate target, one is left wondering about what triggers the production of active cdc2 that initiates this feedback loop in the first place. Clearly, any reaction which downregulates wee1 or upregulates cdc25 could tip the balance toward cdc2 activation. Many candidates for such a mitotic trigger exist. For example, the accumulation of a critical level of cyclin due to new synthesis may be sufficient to trigger mitosis (see discussion in [lo”]). Alternatively, several kinases (e.g. ME kinases, calcium/calmodulin kinase II [CaMKII], or nimA) may be important for mitosis. As discussed above, the ME kinase(s) may modulate cdc2 activity through cdc25 and weel, but this kinase activity also has many other substrates at mitosis. Alternatively, CaMKII may contribute to this G2/M trigger. Fission yeast expressing a constitutively active CaMKII arrest in G2; this process is apparently not mediated through the weel/cdc25 pathway [32]. CaMKII may operate through the nimA kinase, encoded by a gene first described in Aspergillus that is indispensable for mitosis [33]. Finally, the mitotic trigger mechanism may involve some uncharacterized cdc2 inhibitor protein or other novel mechanism. Whether the mitotic trigger is mediated through cdc25, weel, ME kinase, or some undefined enzyme, once the switch is thrown, even a modest amount of active cdc2 would self-catalyze rapid and irreversible mitotic entry (Fig. 3).
Other
cell cycle regulatory
mechanisms
Several observations have suggested that regulatory mechanisms not involving Tyrl5 phosphorylation are important for the mitotic control of cdc2. In fission yeast, mutations that block cdc2 Tyr15 phosphorylation both accelerate entry into mitosis and abolish the dependence of mitosis on DNA replication [34]. In contrast, although budding yeast possess a homologous weel/cdc25 system, mutating the equivalent tyrosine residue causes no deleterious effects (reviewed in [35]). Similarly, deletion of SWE? (the budding yeast wee1 homologue) neither alters cell cycle progression nor
abolishes the DNA-damage checkpoint [36*]. Whether this alternative mode of cell cycle regulation is universal or restricted to budding yeast must await further study A second demonstration of novel cell cycle regulatory mechanisms has been seen during early Drosophila embryogenesis. Edgar et al. [37**] define three embryonic stages with distinct cell-cycle regulation by performing immunoblot analysis on precisely staged individual embryos. During the first stage (cycles 2-7), cdc2-cyclin complexes display no oscillation in cdc2 activity; rather, these cell cycles proceed in the presence of constitutively active cdc2 kinase. The authors propose that localized cyclin B degradation (i.e. of nuclear-associated cyclin B) may explain how cells cycle in the presence of globally active cdc2. During the second stage (cycles 8-13), cyclin-limited cycles occur: cyclin B content does not change appreciably through cycle 7, but around cycle 10 cyclin B oscillations have increased to such an extent that they drive mitosis. During this stage, the cell cycle begins to slow as the synthesis of cyclin B increases. These cyclin-limited cycles appear to proceed in the absence of any inhibitory phosphorylations on cdc2, although the experiments cannot eliminate the possibility of a small subpopulation of cdc2 which is tyrosine phosphorylated [370*]. The absence of inhibitory phosphorylation of cdc2 probably results from the abundance of maternally supplied string (a homologue of cdc25). During the third stage (cycles 14-16), when maternally supplied string is degraded, dephosphorylation of cdc2 becomes rate-limiting [37**]. This last stage, therefore, is regulated by transcription/translation of string. In summary, these novel embryonic M phase control systems indicate that multiple modes of cell cycle regulation can occur in the same model system. Drosophila and Xenopus early cell divisions have modified cell cycles in which S phase and mitosis immediately follow one another without any gap phases. Despite the rapidity of these early cell cycles, these embryos replicate all of their DNA accurately in each cell cycle. Recent work has revealed the existence of a number of small inhibitor proteins which offer a potential mechanism for coupling DNA replication with cell cycle progression. These cyclin kinase inhibitors function during Gl by binding to and inhibiting the catalytic activity of various cdks (see Elledge and Harper, this issue pp 847-852). One such safeguard, which is encoded by an essential maternally derived transcript and functions during early Drosophila development, is the product of the plutonium gene (plu [38]), which encodes a small protein containing ankyrin repeats (protein-protein interaction motifs). A similar small protein (p161NK4) containing multiple ankyrin repeats is present in mammalian cells [39]. It seems plausible that plu, like p161NK4, might regulate a cdk that controls replication [39]. Given the conservation of mechanisms governing all cdks, it seems reasonable to anticipate that similar regulators of cdc2 might exist. By analogy with plu/p16lNK4, these potential cdc2 regulators would drive alternating M phases via their ability to inhibit MPF (cdc2-cyclin B). The exis-
Cdc2 regulatory
tence of such factors would add an additional level of control on cdc2-cyclin activity.
Conclusion
factors Coleman
and Dunphy
a protein Unase that can activate p33rdkz and p34Ck2. EM60 / 1993, 12~3123-3132. This paper demonstrates that immunoprecipitated Xenopus MO1 5 or purified recombinant MO1 5 can phosphorylate the Thrl60 residue of bacterially expressed cdk2, resulting in active cdk2 kinase. The recombinant MO1 5 must be incubated in Xenopus extracts in order to induce its CAK activity. The role of binding partners and phosphorylation in activating MO1 5 are discussed. 7.
During the past year, great strides have been made towards unraveling the complex network of kinases and phosphatases which regulate cdc2 activity and ultimately control progression through the cell cycle. Given that many aspects of the cell cycle remain mysterious, it is likely that novel regulatory pathways and other surprises await discovery. Eventually, through studying the interplay between the various cell cycle modifying enzymes, we will begin to address mndamental issues such as how a cell coordinates its division with intracellular and intercellular cues.
Solomon Ml, Harper JW, Shuttleworth I: CAK, the p344ck2 activating kinase, contains a protein identical ur cl&ly related to p40M0’5. FMBO 1 1993, 12:3133-3142. This group isolates CAK activity from Xenopus egg extracts; micropeptide sequencing and immunodepletion experiments demonstrate that the catalytic subunit of CAK is MO1 5. Implications for the regulation of CAK are discussed.
.
a. ..
Fisher RP, Morgan DO: A novel cyclin associates with MOlS/CDK7 to fomt the CDK-activating kinase. Cell 1994, 78:713-724. CAK activity purified from mammalian cells comprises two major proteins, a 42 kDa homologue of MO1 5 fcdk7) and a 37 kDa cyclin-like protein tcyclin HI. CAK activity can be reconstituted by combining recombinant forms of these proteins in vitro. Mutation of the cdk7 Thrl70 residue (analogous to Thribl in cdc2J results in nearly complete loss of CAK activity. The substrate specificity of CAK toward various cdk-cyclin combinations is discussed. 9.
Acknowledgments
Lee TH, Solomon MJ, Mumby MC, negative regulator of MPF, is a form 2A. Ce// 1991, 64:415423.
Kirschner MW: INH, a of protein phosphatase
Lee TH, Turck C, Kirschner MW: Inhibition of Cdc2 activation by INHIPPZA. MO/ Biol Cell 1994, 5:323-338. zis paper characterizes the mechanism by which PPZA (INH) negatively regulates entry into mitosis. Ultimately, PP2A inhibits the switch in tvrosine kinase and tvrosine ohosohatase activitv that accomoanies mitosis, but its mechan/sm of &ztion may be mediated via the’ cdc2 Thrlbl ohosohorvlation pathway. PPZA inhibits Thrl61 ohosohorvlation and’ thu; may be one of the rate-limiting events during the C;/M transition. 10.
We wish
to thank
the
members
of the
Dunphy
laboratory
and
S
Fashena for critical readings, and M Alvarez for help preparing the manuscript. TR Coleman is supported by a fellowship from the National htiNteS of Health (NRSA); WC Dunphy is an investigator
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DeBondt HL, Rosenblatt J, tancarik J, Jones HD, Morgan DO, S-H: Crystal structure of cyclin-dependent kinase 2. Narure 1993, 363:595-602. The crystal structure ol human cdk2 is presented. This enzyme consists of an amino-terminal lobe (containing the cyclin-binding site) and a larger carboxy-terminal lobe, which together surround the substratebinding cleft. Given the conserved nature of all cdks, this structure has important implications for the molecular details of how cyclin binding and phosphorylation regulate this family of enzymes. Kim
5. .
Fesquet 0, Labbe J-C, Derancourt J, Capony J-P, Galas S, Cirard F, Lorca T, Shuttleworth J, Don.% M, Cavadore J-C: The MO15 gene encodes the catalytic subunit of a protein kinase that activates cdc2 and other cyclindependent kinases fCt3K.s) through phosphorylation of ThrlCl and its homologues. FM80 / 1993, 12:3111-3121. Using starfish oocytes as a source material, this group purified a kinase that activates cdc2 via phosphorylation on the Thr161 residue. Peptide sequencing of the catalytic subunit of this CAK activity revealed that it is the starfish homologue of the MO15 gene.
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18. .
Parker LL, Walter SA, Young PC, Piwnica-Worms rylation and inactivation of the mitotic inhibitor Niml/Cdrl kinase. Ndrure 1993, 363:73&738. See note to [17*1.
H: Phosphowee1 by the
19. Wu L, Russell P: Niml kinase promotes mitosis by inactivating . wee1 tyrosine kinase. Narure 1993, 363:738-741. This paper demonstrates that the level of wee1 phosphorylation is regulated, at least in part, by the level of niml expression in vivo: wee1 is
881
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Cell multiplication hyperphosphorylated rylated in ninil-yeast. wee1 in vitro.
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20. .
Tang 2, Coleman TR, Dunphy WC: Two distinct mechanisms for negative regulation of the wee1 protein kinase. EMBO / 1993, 12:3427-3436. The potential mechanism ofweel regulation is examined by adding yeast wee1 to Xenopus extracts. The added wee1 oscillates between an underphospholylated form during interphase and a hyperphosphorylared form at mitosis. The M phase hypetphosphorylated form results in greatly reduced cdc2-specific tyrosine kinase activity. Hyperphosphotylation of wee1 appears to occur in the absence of the cdc2 kinase. 21.
Kuang J, Zhao J-Y, Wright DA, Saunders GF, Rao PN: Mitosisswcific monoclooal antibody MPM-2 inhibits Xenopus oocyte &turation and depletes r&ration-promoting a&v@. &c Nat\ Acad Sci USA 1989, 86:49824986.
22.
Mutations in cdc25 that remove conserved cdc2 phosphorylation sites decrease phosphorylation and activation by cdc2. Unlike wild-type controls, these mutations fail to activate pre-MPF in oocyte extracts. These findings implicate a positive-feedback loop between cdc2 and cdc25. 31. .
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C, Karsenti E: Dephosphotyprotein phosphatase: specific in Xenopus egg extracts. MO/
sites MO/
of fission yeast cell cycle control of mitosis on DNA replication.
regulatory motif of cyclinD/CDK4.
in
TR Coleman and WC Dunphy, Howard Hughes Medical Institute, Division ofBiology 216-76, California Institute of Technology, Pasadena, CA 91125, USA.
factors
Thomas R Coleman and William G Dunphy California
Institute of Technology,
Pasadena,
USA
A growing family of kinases and phosphatases controls the activity of the cyclin-dependent kinase cdc2. The past year has seen the identification of the cdk activating kinase as well as considerable elucidation of the cdc25/weel regulatory pathways. Both cdc25 and wee1 appear to be regulated by upstream kinase/phosphatase networks. In addition, it is likely that other regulatory mechanisms cooperate with the weel/cdc25 phosphorylation systems to control the action of cdc2. Together, these elaborate checks and balances ensure that cdc2 triggers mitosis at the appropriate time. Current
Opinion
in Cell Biology
1994,
6:877-882
an enzyme known as cdk activating kinase (CAK). In contrast, phosphorylation on either Thrl4 or Tyr15 by multiple weel-like kinases dominantly inhibits cdc2 activity. At the onset of mitosis, the cdc25 phosphatase activates cdc2 by dephosphorylating Tyr15 and most probably Thrl4. Exit from mitosis occurs with degradation of the cyclin, dephosphorylation of Thrl61, and consequent inactivation of MPF (Fig. 1). This review focuses mainly on the regulation of cdc25 and weel.
Introduction In all eukaryotes, cyclin-dependent kinases (cdks) regulate each phase of the cell cycle (for background reviews, see [l-3]). The first discovered cdk, the cell division cycle 2 (cdc2) kinase, serves as the catalytic component of maturation promoting factor (MPF), a dominant regulatory factor that initiates mitosis by phosphorylating a wide variety of structural and regulatory proteins. Control of the cdc2 kinase is a highly regulated process which requires binding to a regulatory subunit (cyclin B) and concomitant phosphorylation at multiple, highly conserved residues. Phosphorylation on Thrl61, which is necessary for cdc2 kinase activation, is mediated by
CAK: the cdc2 activating
kinase
Activation of the cdc2-cyclin CAK-mediated phosphorylation
heterodimer requires of Thrl61. The crys-
Interphase Thr 161
Thr 14
Interphase
Tyr 15
rl
rl
cdc2
cdc2 cdc25
Dephosphorylation
,
3
k%Y m
I
Kinases
I
cyclin I
-----,
r----I I
I
cyclin I I I I I _ - - - - - - - - - _’ Degradation Active
MPF
8 1994 Current Opinion in Cell Biology
Fig. 1. Cdc2 and cyclin B control entry into mitosis. During interphase, newly synthesized cyclin B binds to cdc2 inducing its phosphorylation on three conserved residues. CAK activates cdc2 by phosphorylating the Thrl61 site; concomitantly, several Thrl4/TyrlS kinases dominantly inhibit cdc2 by phosphorylating the Thr14 and Tyrl5 sites. The cdc25 phosphatase induces mitosis by dephosphorylating these inhibitory phosphates, producing active MPF. Exil from mitosis occurs with degradation of cyclin and dephosphorylation of Thrl61 by an unknown phosphatase.
Abbreviations CAK-cdk activating MPF-maturation
kinase; CaMKlI-caIcium/calmoduIin promoting factor; MPM-2-mitosis
0 Current
kinase II; cdkprotein monoclonal
Biology
cyclin-dependent kinase; antibody-2; PPZA-protein
ltd ISSN 0955-0674
ME-MPM-2 phosphatase
epitope; 2A. 877
878
Cell multiplication
tal structure of human cdk2 reveals that the Thr160 residue (analogous to Thr161 of cdc2) resides on a structural loop that occludes the substrate-binding cleft. Thus, phosphorylation on this residue may alter the geometry of this inhibitory loop and stabilize an active conformation [4**]. Three groups [5*-7.1 determined that CAK is composed of a complex of at least one regulatory subunit (p37) and a catalytic subunit (M015). Interestingly, MO15 is structurally related to the cdc2 kinase, and the p37 regulatory subunit shares limited homology with the cyclin family. Together these two components are suffrcient to reconstitute CAK activity in a highly purified system [8**]. A proposal to rename MO15 as cdk7 and its p37 binding partner as cyclin H has been suggested [W]. The regulation of Thr161 phosphorylation has been a matter of widespread interest. Lee and co-workers [9] initially proposed that an inhibitor of MPF activity directly opposed the action of CAK by dephosphorylating Thr161. More recent data from this group, however, show that this inhibitor is a conventional form of protein phosphatase 2A (PP2A) that inhibits MPF by a less direct means. The addition of exogenous PP2A to Xenop~rs egg extracts inhibits Thrl61 phosphorylation in a dose-dependent manner during the lag period before mitosis, when the pivotal reaction(s) that trigger mitosis are expected to occur [lO**]. These findings suggest that PP2A may act indirectly on CAK or the Thrl61directed phosphatase (Fig. 2). This proposal, however, is apparently at odds with the fact that Thrl61 phosphorylation is constitutive throughout the cell cycle [ll]. Finally, the identity of the phosphatase that dephosphorylates Thr161 is not known, but a recently discovered phosphatase that can bind cdks (named Cdil, Cip2, or KAP [12-141) is a potential candidate.
Membrane-bound 161 14 15
al N
Active
Inactive
wee1
C
wee1
Inactive
wee1
0 1994 Current Opinmn in Cell Biology
Fig. 2. Multiple kinases and phosphatases prepare cdc2 and cyclin for activation. CAK stimulates cdc2 activity by phosphorylating (P) the Thr161 residue of cdc2. Both wee1 and a membrane-bound kinase phosphorylate the inhibitory residues Thrl4 and Tyrl5. Wee1 can be inactivated by several phosphorylation events. The niml kinase mediates one such event by phosphorylating the catalytic (carboxy-terminal, C) domain. An unknown phosphatase opposes niml to produce active weel. Multiple kinases tcdc2 and kinase X) most probably mediate the second inhibitory event by extensively phosphorylating the regulatory (amino-terminal, N) domain. This amino-terminal inhibitory phosphorylation is opposed by a PPZA-like activity.
ulated by some other pathway that operates in parallel with the accumulation of cyclin.
Regulation
of the wee1 protein
kinase
Genetic studies in yeast that demonstrated that the wee1 kinase is negatively regulated by the niml kinase [15,16] were validated by recent biochemical analyses. Specifically, the recombinant niml protein directly phosphorylates and inactivates isolated fission yeast wee1 in vitro [170-l ‘91. Interestingly, this niml-catalyzed phosphorylation of wee1 occurs in the carboxy-terminal region [17*], the area that contains the kinase domain. To date, niml homologues have not been identified in other species, and experimental evidence indicates that Xenopus egg extracts appear to lack a niml-like activity (T Coleman, W Dunphy, unpublished data). Therefore, a general role for niml in the control of wee1 activity remains to be demonstrated. Similarly, the question of what regulates yeast niml activity during the cell cycle remains unanswered. As wee1 is downregulated during mitosis when MPF is activated, MPF is one candidate for a niml activator. Alternatively, niml may be reg-
Whether or not niml homologues are present in other eukaryotes, phosphorylation by niml is not the sole mechanism through which wee1 is regulated. For example, deletion of the yeast nirrr1 gene is not lethal, but rather results in a prolonged Gz phase [15]. Direct evidence demonstrating the existence of a second wee1 regulatory pathway comes 13om a heterologous system in which fission yeast wee1 was introduced into Xerropus egg extracts [20*]. Mitotic, but not interphase, egg extracts contain a kinase which extensively phosphorylates the amino-terminal region of fission yeast weel. Like wee1 that has been phosphorylated in the carboxyterminal domain by niml, the.weel protein phosphorylated in its amino-terminal region is almost completely inactive as a cdc2-specific tyrosine kinase [20*]. An attractive candidate for the weel-inhibitory kinase is MPF Extensive phosphorylation of weel, however, can occur in mitotic extracts depleted of cdc2, suggesting that cdc2 is not the sole kinase responsible for inactivation of wee1 at mitosis [20-l. The phosphatase that serves to main-
Cdc2
regulatory
factors
Coleman
and Dunphy
tain wee1 in its active, underphosphorylated form during interphase possessesseveral hallmarks characteristic of a PP2A-like activity [10**,20*] (Fig. 2).
will therefore be necessary to elucidate the relationship between the replication checkpoint and cdc2 tyrosine phosphorylation.
The more recent characterization of a Xetqns wee1 kinase confirms the existence of a wee1 regulatory kinase/phosphatase pathway in an entirely homologous system (P Mueller, T Coleman, W Dunphy, unpublished data). The activity of endogenous Xerroprrswee1 protein is stringently regulated during the cell cycle: the interphase underphosphorylated form etliciently phosphorylates cdc2 exclusively on Tyrl5. whereas the mitotic hyperphosphorylated form is nearly inactive. Dephosphorylation of the mitotic form by treatment with PP2A restores its kinase activity. Moreover, in vitro treatment of Xcrrtipns wee1 with active cdc2-cyclin complex results in phosphorylation and inactivation ofweel kinase. At least one additional wee1 inhibitory kinase probably exists, however, as Xcnopprrswee1 becomes partially phosphorylated in mitotic extracts that have been depleted of active cdc2. Candidates for this weel-specific kinase include the MPM-2 epitope (ME) kinases that generate phosphopeptide epitopes on a large number of proteins recognized by the mitosis protein monoclonal antibody, MPM-2. The identities of the ME kinases have not been definitively established (21-231 but evidence suggests that ME kinase activity phosphorylates both fission yeast and Xenopns wee1 during mitosis (P Mueller, T Coleman, W Dunphy, unpublished data).
In vertebrates, cdc2 is also phosphorylated on Thrl4, the residue that adjoins Tyrl5, but the identity of the inhibitory kinase that phosphorylates cdc2 on this residue remains unclear. Interestingly, Kornbluth et al. [25*] reported a membrane-associated kinase activity that can phosphorylate Thr14 (a co-fractionating activity also phosphorylates Tyr15). It will be interesting to learn in which cellular membrane this kinase resides. Specific membrane localization may target the kinase to certain cdc2-cyclin subpopulations, allowing for differential activation/inactivation of MPF within the cell.
Another issue in wee1 regulation concerns the replication checkpoint. In Xmop~ts egg extracts, incomplete DNA replication blocks the activation of cdc2 by maintaining it in a tyrosine-phosphorylated form. It has been proposed that the control system that monitors DNA replication acts through a cdc2-specific tyrosine kinase [24]. In direct measurements, however, the activity of Xewpus wee1 does not appear to be modulated by the presence of unreplicated DNA (P Mueller, T Coleman, W Dunphy, unpublished data). Further studies
Inactive
Regulation
of
the cdc25 phosphatase
It is now well established that the cdc25 phosphatase is weakly active during interphase and fully active during mitosis [26,27]. Moreover, cdc25 activity is regulated by a kinase/phosphatase system: the active form has an extensively phosphorylated amino-terminal domain. As in the wee1 regulatory pathway, the cdc25 regulatory phosphatase appears to be a PP2A-like protein (Fig. 3). Furthermore, cdc25 can be activated following in vitro phosphorylation by active cdc2-cyclin complex [28,29]; the phosphorylation sites are consistent with those characterized in viva on mitotically active cdc25 [29,30-l. Interestingly, Xenopus extracts deficient in cyclin B can also phosphorylate cdc25 in the presence of the phosphatase inhibitor okadaic acid [27]: these results suggest the presence of additional cdc25 kinases. Likewise, Kuang and co-workers [31*] have shown that cdc25 is a major substrate of the ME kinase at mitosis. Thus a variety of kinases, including cdc2 and ME kinase(s), may contribute to cdc25 regulation, as is the case for the wee1 protein.
cdc25
Nuclear
envelope
Spindle
apparatus
Chromosomal
DNA
8 1994 Curmnl Gpinion in Cell Biology
Fig. 3. At the Gz/M transition, a positive feedback loop activates the cdc25 phosphatase and inactivates wee1 resulting in abrupt entry into mitosis. Inactive triplyphosphorylated cdc2-cyclin complexes can be activated by dephosphorylating the Thrl4 and Tyr15 residues, a process mediated by active cdc25 and opposed by the wee1 kinase. The cdc25 phosphatase oscillates between an amino-terminally phosphorylated (active) form and an underphosphorylated (inactive) form. Cdc25 activity is opposed by a PPZA-like activity and favored by an unknown kinase X fan ME kinase?) and MPF, which also drives this reaction forward by phosphorylating and inactivating the wee1 kinase. MPF promotes mitosis by acting on the nuclear envelope, the spindle apparatus and the chromosomal DNA.
879
;80
Cell multiplication
Many striking parallels exist between the regulation of wee1 and that of cdc25. Both proteins are’extensively phosphorylated during the G2/M transition. Phosphorylation of wee1 abolishes its kinase activity, while phosphorylation of cdc25 augments its phosphatase activity. As both proteins are phosphorylated in parallel, they may be controlled by the same kinase/phosphatase system. Indeed, both wee1 and cdc25 are dephosphorylated by a PP2A-like activity, and the cdc25-specific PP2Alike enzyme appears to be less active during mitosis [27]. Similarly, both proteins are substrates for the kinase activity that they control (MPF), thus suggesting an appealing positive-feedback loop wherein a small amount of active cdc2 would rapidly and irreversibly trigger mitosis (Fig. 3). Because the activities of cdc25 and wee1 are quite probably regulated by their ultimate target, one is left wondering about what triggers the production of active cdc2 that initiates this feedback loop in the first place. Clearly, any reaction which downregulates wee1 or upregulates cdc25 could tip the balance toward cdc2 activation. Many candidates for such a mitotic trigger exist. For example, the accumulation of a critical level of cyclin due to new synthesis may be sufficient to trigger mitosis (see discussion in [lo”]). Alternatively, several kinases (e.g. ME kinases, calcium/calmodulin kinase II [CaMKII], or nimA) may be important for mitosis. As discussed above, the ME kinase(s) may modulate cdc2 activity through cdc25 and weel, but this kinase activity also has many other substrates at mitosis. Alternatively, CaMKII may contribute to this G2/M trigger. Fission yeast expressing a constitutively active CaMKII arrest in G2; this process is apparently not mediated through the weel/cdc25 pathway [32]. CaMKII may operate through the nimA kinase, encoded by a gene first described in Aspergillus that is indispensable for mitosis [33]. Finally, the mitotic trigger mechanism may involve some uncharacterized cdc2 inhibitor protein or other novel mechanism. Whether the mitotic trigger is mediated through cdc25, weel, ME kinase, or some undefined enzyme, once the switch is thrown, even a modest amount of active cdc2 would self-catalyze rapid and irreversible mitotic entry (Fig. 3).
Other
cell cycle regulatory
mechanisms
Several observations have suggested that regulatory mechanisms not involving Tyrl5 phosphorylation are important for the mitotic control of cdc2. In fission yeast, mutations that block cdc2 Tyr15 phosphorylation both accelerate entry into mitosis and abolish the dependence of mitosis on DNA replication [34]. In contrast, although budding yeast possess a homologous weel/cdc25 system, mutating the equivalent tyrosine residue causes no deleterious effects (reviewed in [35]). Similarly, deletion of SWE? (the budding yeast wee1 homologue) neither alters cell cycle progression nor
abolishes the DNA-damage checkpoint [36*]. Whether this alternative mode of cell cycle regulation is universal or restricted to budding yeast must await further study A second demonstration of novel cell cycle regulatory mechanisms has been seen during early Drosophila embryogenesis. Edgar et al. [37**] define three embryonic stages with distinct cell-cycle regulation by performing immunoblot analysis on precisely staged individual embryos. During the first stage (cycles 2-7), cdc2-cyclin complexes display no oscillation in cdc2 activity; rather, these cell cycles proceed in the presence of constitutively active cdc2 kinase. The authors propose that localized cyclin B degradation (i.e. of nuclear-associated cyclin B) may explain how cells cycle in the presence of globally active cdc2. During the second stage (cycles 8-13), cyclin-limited cycles occur: cyclin B content does not change appreciably through cycle 7, but around cycle 10 cyclin B oscillations have increased to such an extent that they drive mitosis. During this stage, the cell cycle begins to slow as the synthesis of cyclin B increases. These cyclin-limited cycles appear to proceed in the absence of any inhibitory phosphorylations on cdc2, although the experiments cannot eliminate the possibility of a small subpopulation of cdc2 which is tyrosine phosphorylated [370*]. The absence of inhibitory phosphorylation of cdc2 probably results from the abundance of maternally supplied string (a homologue of cdc25). During the third stage (cycles 14-16), when maternally supplied string is degraded, dephosphorylation of cdc2 becomes rate-limiting [37**]. This last stage, therefore, is regulated by transcription/translation of string. In summary, these novel embryonic M phase control systems indicate that multiple modes of cell cycle regulation can occur in the same model system. Drosophila and Xenopus early cell divisions have modified cell cycles in which S phase and mitosis immediately follow one another without any gap phases. Despite the rapidity of these early cell cycles, these embryos replicate all of their DNA accurately in each cell cycle. Recent work has revealed the existence of a number of small inhibitor proteins which offer a potential mechanism for coupling DNA replication with cell cycle progression. These cyclin kinase inhibitors function during Gl by binding to and inhibiting the catalytic activity of various cdks (see Elledge and Harper, this issue pp 847-852). One such safeguard, which is encoded by an essential maternally derived transcript and functions during early Drosophila development, is the product of the plutonium gene (plu [38]), which encodes a small protein containing ankyrin repeats (protein-protein interaction motifs). A similar small protein (p161NK4) containing multiple ankyrin repeats is present in mammalian cells [39]. It seems plausible that plu, like p161NK4, might regulate a cdk that controls replication [39]. Given the conservation of mechanisms governing all cdks, it seems reasonable to anticipate that similar regulators of cdc2 might exist. By analogy with plu/p16lNK4, these potential cdc2 regulators would drive alternating M phases via their ability to inhibit MPF (cdc2-cyclin B). The exis-
Cdc2 regulatory
tence of such factors would add an additional level of control on cdc2-cyclin activity.
Conclusion
factors Coleman
and Dunphy
a protein Unase that can activate p33rdkz and p34Ck2. EM60 / 1993, 12~3123-3132. This paper demonstrates that immunoprecipitated Xenopus MO1 5 or purified recombinant MO1 5 can phosphorylate the Thrl60 residue of bacterially expressed cdk2, resulting in active cdk2 kinase. The recombinant MO1 5 must be incubated in Xenopus extracts in order to induce its CAK activity. The role of binding partners and phosphorylation in activating MO1 5 are discussed. 7.
During the past year, great strides have been made towards unraveling the complex network of kinases and phosphatases which regulate cdc2 activity and ultimately control progression through the cell cycle. Given that many aspects of the cell cycle remain mysterious, it is likely that novel regulatory pathways and other surprises await discovery. Eventually, through studying the interplay between the various cell cycle modifying enzymes, we will begin to address mndamental issues such as how a cell coordinates its division with intracellular and intercellular cues.
Solomon Ml, Harper JW, Shuttleworth I: CAK, the p344ck2 activating kinase, contains a protein identical ur cl&ly related to p40M0’5. FMBO 1 1993, 12:3133-3142. This group isolates CAK activity from Xenopus egg extracts; micropeptide sequencing and immunodepletion experiments demonstrate that the catalytic subunit of CAK is MO1 5. Implications for the regulation of CAK are discussed.
.
a. ..
Fisher RP, Morgan DO: A novel cyclin associates with MOlS/CDK7 to fomt the CDK-activating kinase. Cell 1994, 78:713-724. CAK activity purified from mammalian cells comprises two major proteins, a 42 kDa homologue of MO1 5 fcdk7) and a 37 kDa cyclin-like protein tcyclin HI. CAK activity can be reconstituted by combining recombinant forms of these proteins in vitro. Mutation of the cdk7 Thrl70 residue (analogous to Thribl in cdc2J results in nearly complete loss of CAK activity. The substrate specificity of CAK toward various cdk-cyclin combinations is discussed. 9.
Acknowledgments
Lee TH, Solomon MJ, Mumby MC, negative regulator of MPF, is a form 2A. Ce// 1991, 64:415423.
Kirschner MW: INH, a of protein phosphatase
Lee TH, Turck C, Kirschner MW: Inhibition of Cdc2 activation by INHIPPZA. MO/ Biol Cell 1994, 5:323-338. zis paper characterizes the mechanism by which PPZA (INH) negatively regulates entry into mitosis. Ultimately, PP2A inhibits the switch in tvrosine kinase and tvrosine ohosohatase activitv that accomoanies mitosis, but its mechan/sm of &ztion may be mediated via the’ cdc2 Thrlbl ohosohorvlation pathway. PPZA inhibits Thrl61 ohosohorvlation and’ thu; may be one of the rate-limiting events during the C;/M transition. 10.
We wish
to thank
the
members
of the
Dunphy
laboratory
and
S
Fashena for critical readings, and M Alvarez for help preparing the manuscript. TR Coleman is supported by a fellowship from the National htiNteS of Health (NRSA); WC Dunphy is an investigator
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regulatory motif of cyclinD/CDK4.
in
TR Coleman and WC Dunphy, Howard Hughes Medical Institute, Division ofBiology 216-76, California Institute of Technology, Pasadena, CA 91125, USA.