- Email: [email protected]
The decision to enter mitosis
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The decision to enter mitosis
The phosphotyrosine content of the cdc2 protein kinase, the catalytic component o[ maturation-promoting [actor OVIPF),is an important parameter o[ mitotic regulation in a variety of organisms. Recent studies have shed considerable light on how the cdc2-specific tyroshle kinase (wee1) and its competing phosphatase (cdc25) are regulated during the cell cycle. A goal [or the Future will be to obtain a comprehensive picture of how the wee1--cdc25 regulatory system collaborates with other steps in mitotic activation to ensure that cell division occurs at the appropriate tiine during the cell cycle.
WilliamG, Dunphyis at the Divisionof Biology 216-76, California Instituteof Technology, Pasadena,CA 91125, USA,
In all eukaryotes, a variety of cyclin-dependent prorein klnases drive the Initiation and progression of each successive phase of the cell cycle t. The onset of mitosis Is coupled closely to the activation of MPF, a heterodimerlc complex that contains a B-type cyciln and the cdc2 protein kinase ~4. Except for its destruction at the metaphase-anaphase transition s, rela-
tively little Is known about post-translational regu. latlon of the cyclln subunlt of MPF. By contrast, a
OAK
great deal is known about the molecular control of cdc2 catalytic activity prior to cell division. Several cdc2-specific stimulatory and inhibitory kinases play opposing roles in the cdc2 activation process (Fig. 1). A stimulatory kinase known as cdk-activating kinase (CAK) phosphorylates Thr 161, thereby allowing cdc2 to phosphorylate its substrates 6-8. Conversely, an inhibitory kinase (weed phosphorylates cdc2 on Tyrl5, thus impeding its activity9-~. It is also thought that a distinct inhibitory kinase 12,13collaborates with weel by phosphorylating cdc2 on an adjacent residue (Thrl4). One of the final events in the initiation of mitosis is the tyrosine dephosphorylation of cdc2 by the cdc25 protein, an extremely specific and highly regulated phosphatase 1.-)7. In fission yeast, competition between the cdc25 and weel proteins appears to be a critical determinant of mitotic timing t. For this reason, there has been considerable interest in how the actions of these proteins are regulated during the cell cycle. Recent studies indicate that the catalytic activities of both the cdc25 and weel proteins are controlled tightly by an upstream network of kinases and phosphatases ls-z2. However, it is not entirely clear how these phosphorylation reactions, which ultimately regulate the phosphotyrosine content of cdc2, contribute to the initial decision to enter mitosis. Another issue is that the relative importance of tyrosine phosphorylation as a cdc2-regulatory mechanism appears to vary considerably depending upon the particular characteristics of the cell cycle tn a given organism z3.z7. This apparent lack of universality raises an important question: does the underlying mechanism for controlling the Initiation of mitosis differ among species or Is the basic mechanism conserved, with refinements to accommodate the needs of particular species? The widespread interest In this question has been heightened by the likely possibility that the mitotic decision mechanism is controlled by cell cycle checkpoint factors, the proreins that render the Initiation of mitosis dependent upon the successful completion of earlier cell cycle processes such as accurate DNA replication 2~,z9,
cdo25
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1
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Mitosis
niml FIGURE 1 Post-translational regulation of the cdc2-cyclin.B complex. Cdk-activating kinase (CAK) stimulates cdc2 activity by phosphorylating Thr161. The cdc25, wee1 and niml proteins cont,ol the inhibitory tyrosine phosphorylation of cdc2 on Tyrl S. 202
© 1994ElsevierScienceLtd 0962-89241941507.00
Regulation of the cdc2S protein by phosphorylatlon reactions It is well established that the cdc25 protein is weakly active during interphase and becomes fully active at the G2-M transition owing to an extensive phosphorylation of its N-terminal regulatory domain at approximately six sites 19,2".a~z. During interphase, the cdc2S.stimulatory kinase is weakly active, and a potent cdc25.inhibitory phosphatase opposes its action (Fig. 2). This inhibitory phosphatase is quite sensitive to okadaic acid and has several characteristics reminiscent of protein phosphatase 2A (PP2A). Both the cdc25-regulatory enzymes are tightly regulated in a manner consistent with their role in controlling the activation of the cdc25 protein: at the G2-M transition, the activities of the cdc25-stimulatory kinase and cdc25-inhibitory phosphatase increase and decrease, respectively ~9. The identity of the cdc25-stimulatow kinase has been examined in a number of studies, cdc2--cyclin-B TRENDSIN CELLBIOLOGYVOL, 4 JUNE1994
i can phosphorylate cdc25 well/n vitro3~.3z. However, Xenopus extracts containing little or no cyclin B can effect the phosphorylation of cdc25 efficiently once PP2A action is blocked by okadaic acid ~9, The recent studies of Kuang et al. 3~may help to clarify this issue. They demonstrated that the cdc25 protein is a substrate of the ME (MPM-2 epitope) kinase, the enzyme responsible i¢,r the creation of a phosphopeptide epitope in a myriad of mitotic phosphoproteins recognized by the MPM-2 monoclonal antibody. One possibility is that the ME kinase, cdc2 and perhaps other kinases all contribute to the activation of cdc25. In this regard, an important issue is whether the ME kinase is regulated by cdc2 or is controlled independently. In the former case, the ME kinase would serve as an intermediary in the cdc2-dependent, self-perpetuating activation of cdc25, whereas in the latter case the activation of the cdc25 protein could be modulated by events that precede the switching-on of the cdc2 protein. This latter possibility is especially intriguing in view of the fact that biochemical activities in Xelzopus egg extracts distinct from cdc2-cyclin-B appear to be able to act as MPF under certain circumstances 34-:~6.
Regulation of the wee1 protein by multiple klnases Not surprisingly, in vitro experiments have suggested the activity of the weel proteln is highly regulated during the cell cycle. The first clue about wee1 regulation was provided by genetic experiments that identified the niml protein kinase as a negative regulator of the weel pathway in fission yeast:~7,:~8.These studies were validated by the demonstration that recombinant niml protein can phosphorylate and completely inactivate the isolated wee] protein in a cell-free reaction :¢~4=. A notable feature of the nlmlcatalysed phosphowlation of the weel protein Is that It is restricted to the C.terrnlnal region of the protein that forms its kinase domain, suggesting a potentially direct effect on catalytic function, in fission yeast, the action of the niml protein is involved in the nutritional regulation of mitotic timing :.7,:~. At present, a niml homologue from higher somatic cells has not been described, and Xe,opus egg extracts appear to lack a niml-like activity, as judged by biochemical assays. For these reasons, it remains to be established whether phosphorylation of the catalytic domain of the weel protein by niml-like kinases is a widely used mechanism for mitotic regulation in higher eukaryotic cells where physiological parameters other than nutrient availability are more crucial. Although the niml protein is clearly able to modulate the activity of the weel protein over a wide range, a variety of arguments suggested that it would not be the only regulator of weel. Indeed, mitotic Xenopus egg extracts were shown to contain a kinase activity specific for the N-terminal region of recombinant fission yeast weel, which effected an -60 kDa increase in the apparent molecular mass of weel on SDS-gei electrophoresis 4z. This extensive phosphoryiation, like the more modest pho.~phorylation catalysed by the niml protein, leads to the almost complete inactivation of the ability of weel to
TRENDSIN CELLBIOLO~3YVOL. 4 IUNE 1994
cdc25-stimulatory kinase(s)
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cdc25 PP2A
mitosis
interphase
FIGURE2 Regulation of cdc25 activity.The phosphatase activityof the cdc25 protein is modulated by both a cdc25-stimulatory kinase that phosphorylates its N-terminal, regulatory domain and a cdc25-inhibitory phosphatase (shown here as PP2A)that counteracts the activation process. phosphorylate cdc2. The identity of the weelinhibitory kinase(s) in these in vitro experiments has not been established with certainty, but cdc2depleted extracts maintained in M phase with phos. phatase inhibitors contain high levels of the activity. The inhibitory phosphorylation of the N-terminal region of the weel protein is counteracted by an okadaic-acid-sensitive phosphatase (PP2A-like) that is highly active in interphase Xe,opus egg extracts and thus maintains weeI in its active form. Since it is not yet established whether somatic cells such as fission yeast contain the N-terminal-specific, weelinhibitory kinase, it is not known how this activity and the him 1 protein kinase might collaborate with one another in weel regulation (Fig. 3).
Coordination of cdc25 and wee1 regulation There are some striking parallels between the regulation of cdc25 and weel. Both proteins are
low activity
high activity
weel-lnhibitory klnase(s)
wee1
.,= PP2A
n''lT?
Y
low activity FIGURE3 Two kinase-phosphatasesystemsregulatewee1 activity. The niml protein and a competing phosphatasecontrol the phosphory!ationof the catalyticdomain of the wee1 protein. In addition, a distinct kinaseactivity can shutoff wee1 by phosphorylatingits N-terminaldomain, and is counteractedby a PP2A.qke phosphatase. 203
III III I I II
I I
I
NI
Ic
cdc2 does not seem to serve as a potent catalyst for its own activation 46,47. It seems that there is more of the mitotic puzzle to be deciphered.
cdc25
I I
111 II III I
I IIIII
II
"1
•
II
Ic
wee1 FIGURE 4 Potential phosphorylation sites in the XenopusC-type cdc25 protein (see Ref. 19) and the fission yeast wee1 protein (~ee Ref. 9). The location of Ser/Thr-Pro motifs (14 in total for cdc25 and 25 in total for wee1 ) are indicated with a bar. In addition, the locations of catalytically critical residues (Cys for cdc2S and Lys for wee1) are shown.
extensively phosphorylated at the G2-M transition on N-terminal regulatory domains that contain a high density of Ser/Thr-Pro motifs that are potential substrates for a variety of mitotic kinases (Fig. 4). The mechanism by which this phosphorylation influences the function of either the cdc25 or weel protein is not known, in the case of the cdc25 protein, the phosphorylation could either enhance the recognition of the cdc2-cyclin-B complex or relieve a potentially inhibitory effect of the N-terminal domain on the Cterminal catalytic region. Conversely, phosphorylation of the weel protein might either hinder the recognition of the cdc2--cyclin.B complex or result in the inhibition of the C-terminal kinase domain. In addition, the possibility that the phosphorylation of the cdc25 and wee1 proteins might have additional effects on other processes such as intracellular localIzation cannot be excluded. For both the cdc25 and weel proteins, the upstream klnase=phosphatase system Is balanced such that during Interphase both cdc2S and weel remain In an underphosphorylated state, corre. spondlng to their low. and hlgh-actlvlty versions, respectively. A PP2A-Ilke phosphatase has been implicated in both cdc25 and weel regulation, rals. Ing the possibility that the same enzyme might inhibit cdc25 and stimulate weel during inter. phase tg,3°,42-4s. There is evidence that the cdc25inhibitory, PP2A-like phosphatase is downregulated at mitosis tg. Similarly, total kinase activity towards both cdc2S and weel Is greatly elevated at mitosis, the period when cdc2 is most active. Such phosphorylation.-dephosphorylatlon circuits would appear to guarantee the rapid and synchronous (i,e. self-perpetuating) activation of cdc2 at the G2-M transition. However, this scheme suggests a paradox in that the activation of cdc2 requires prior activation of cdc25 and/or inactivation of weel, which in turn would be dependent upon active cdc2. The question arises as to what molecular event tips the balance to favour production of the active cdc2 that would set this activation process in motion. In principle, any factor that could stimulate cdc2S or inhibit weel might be a candidate. However, this triggering event need not involve tyrosine phosphorylation directly. Another consideration is that a low level of active 204
Intracellular localization of cdc2 and its regulators at the G 2 - M transition
The role of intracellular localization in cdc2 regulation is attracting increasing interest. The entry of cdc2-cyclin-B into the nucleus near the time of prophase is well established4s-s°, but the precise role of nuclear translocation in controlling both the actiration and action of the cdc2 protein remains to be determined. The regulators of cdc2 (e.g. cdc25 and weel) likewise display suggestive intracellular locations during the cell cycle. Most investigators appear to concur that the cdc25 protein is largely nuclear in the period just before mitosissl-s4. However, during the remainder of interphase, the cdc2S protein has been observed in either the cytoplasm or the nucleus depending upon the experimental system. For this reason, it is unclear whether the regulated entry of cdc25 into the nucleus is a general feature of mitotic control. Recently, studies on the intracellular location of weel have provided a potentially important perspective on cell cycle regulation. Heald et al. s4 transfected baby hamster kidney (BHK) cells with a gene encoding the human weel-like tyrosine kinase and assessed both the intracellular localization of the expressed protein and its effect on the activity of cdc2-cyclin.B (whose expression was also directed by transfected plasmids). The transfected weel protein was ahnost exclusively nuclear, and the cdc2-cyclinB complex in tile nucleus was apparently inactive, as evidenced by the absence of nuclear mitotic events such as chromosome condensation. By contrast, cytosolic cdc2-cyclin-B remained active as Judged by its ability to induce the depolymerization of a recombinant lamln protein containing a mutation that forced it to remain outside the nucleus. The interesting conclusion posed by these studies is that the activity of the cdt.2-cyclln-B complex may be regulated at least in part by its location in the cell, thus adding a layer of complexity to mitotic regulatory mechanisms. These studies also raise some interesting questions. It might be expected that cytosollc cdc2-cyclln-B is normally under some form of regulation. Is there a distinct cytosollc weel protein or do other regulatory mechanisms not involving tyroslne phosphorylation predominate in the cytoplasm? Role of cdc25 and wee1 proteins In cell cycle checkpoints
in 1989, Hartwell and Welnert introduced the concept of checkpoints to cell cycle control 2x. A checkpoint refers to the process whereby a cell can undergo an arrest or delay of cell cycle progression in response to the perturbation of a cellular process that might ultimately interfere with the successful production of two viable daughter cells, Well-known checkpoints include the block to mitosis in the presence of replicating DNA, the arrest of the cell cycle at any one of several points after DNA damage, and the delay of anaphase in response to the improper association of TRENDS IN CELL BIOLOGY VOL. 4 JUNE 1994
I the chromosomes with the mitotic spindle (see Ref. 29 for a review of checkpoints). In the case of mitotic control, the mechanisms underlying the checkpoint that imposes a G2-1ike arrest of the cell cycle in the presence of unreplicated or damaged DNA have been studied in several experimental systems. In fission yeast, a growing number of genes (e.g. tad1, rad3, radO, rad17 and husl) have been implicated in this process ss-s7. A similar class of genes has been defined in budding yeastsS. An important question is how these gene products impinge upon the activation and/or action of MPF at the G2-M transition. In fission yeast, the cdc2S and weel proteins are clearly involved in a checkpoint-like mechanism that controls the size at which cell division occurs. Consequently, the question arose as to whether the cdc2-regulatory tyrosine kinase-phosphatase system participates in other checkpoints such as those involving unreplicated and/or damaged DNA. The observation that strong overexpression of the cdc25 protein in fission yeast disrupted the replication checkpoint was broadly consistent with this notion so. However, the overexpression of cdc25 did not abolish the DNA-damage checkpoint, suggesting that the replication and damage checkpoints utilize different mechanistic pathways. Moreover, further analysis6° indicated that although cdc25 can influence the replication checkpoint under certain circumstances it appears not to be the pivotal enzyme in the process: for example, a certain weel;cdc25 double mutant strain that is viabl ~. without producing the cdc25 protein can arrest normally in the presence of a DNA synthesis inhibitor (e.g. hydroxyurea). Similarly, the steady-state activity of the cdc25 protein in lnterphase Xcnopus egg extracts is unaffected by the presence of unrepllcated DNA ~'~,2°. The role of the weel protein in the replication checkpoint has also been examined in detail. Fission yeast mutants lacking the activity of either weel or mikl (a related klnase that appears to have a similar function to weel) reportedly both delay mitosis In the presence of hydroxyurea, but a double mutant lacking both weel and mikl activities is profoundly deficient in the replication checkpoint ~:4.sT.so. This observation is consistent with the fact that a fission yeast strain harbouring a mutant version of cdc2 that cannot undergo tyrosine phosphorylation on residue 1S is also deficient in the replication checkpoint 6°. Moreover, experiments in Xenopus egg extracts revealed that the inhibition of DNA replication leads to an elevation of total tyrosine kinase activity specific for cdc22~.6L Nonetheless, in both the fission yeast and frog systems, it has not yet been demonstrated whether the regulation of tyrosine kinase action specific for cdc2 is a direct consequence of the monitoring of unreplicated or replicating DNA. This information will aid understanding of cell cycles in other systems where the tyrosine phosphorylation of cdc2 appears to be less critical. Mitotic regulation without phosphotyroslne
There is now ample precedent for proper mitotic regulation in the absence of apparent tyrosine TRENDSIN CELLBIOLOGYVOL. 4 JUNE1994
phosphorylation on the cdc2 protein. Perhaps the first example described was the early Xenopus embryo, where tyrosine phosphorylation of cdc2 is not detectable during cleavages 2-12 fret. 62). However, the definitive demonstration was provided in the budding yeast where it was shown that strains harbouring an altered cdc2 (CDC28) protein that could not be phosphorylated on the equivalent of Tyrl5 (as well as the preceding threonine residue) could nonetheless grow and divide normally26.27. Strikingly, the replication and repair checkpoints were also fully operational in these mutant strains. These observations have been validated by genetic analysis of the budding yeast weel homologue called SWE163: budding yeast lacking the SWE1 protein divide normally and still can delay mitosis in response to DNA damage. In this regard, it is intriguing that budding yeast do contain an operative weel--cdc2S system (i.e. the SWE1 protein and the cdc25 homologue MIH1; see Ref. 64). One notion is that the function of this system has become iargely cryptic in an organism such as budding yeast that does not rely on a G2 size control in its budding mode of division. Alternatively, the budding yeast weel--cdc25 system may be keyed into physiological parameters that are not readily apparent. An important question is whether mitotic control systems that can operate properly without tyrosine phosphorylation of cdc2 represent special cases or instead reveal a more universal mode of G2-M regulation.
Other mechanismsfor mitotic regulation Although this article has focused heavily on the cdc25 and weel proteins, there are other factors that contribute to mitotic regulation. For exalnple, the phosphorylation of cdc2 on Thr161 is an important prerequisite for its catalytic activity. The catalytic subunit of CAK has recently been identified as the Me15 klnasec'~s. These studies presented data that CAK may possess a partner analogous to a cyciln, and it Is possible that CAK itself is also regulated by phosphorylatlon. In view of this potential complexity, the regulation of Thrl61 phosphorylation by CAK or its competing phosphatase would be a logical target for mitotic control mechanisms, but the evidence to date suggests that the level of Thrl61 phosphorylation is constitutive dmmg the cell cycle6s. During the G1 phase of the cell cycle, a number of small inhibitor proteins, the cyclin kinase inhibitors (CKIs), can bind to and inhibit the catalytic activity of various G l-specific cdks such as cdk2-cyclin-E (see Refs 66 and 67 for reviews). Analogous proteins have not been reported to interact with cdc2 during G2, but the existence of such factors could resolve some of the apparent paradoxes about G2-M regulation. Parenthetically, it is well known that the fission yeast sucl protein (13 kDa) and homologous proteins in other species bind efficiently to cdc2, but these polypeptides appear not to inhibit the catalytic activity" of cdc2, and there is no clear agreement about their involvement in other steps of cdc2 biochemistry~'~. The studies of cdc2--cyclin-B regulation in budding yeast arrested at the replication checkpoint by 20S
treatment with hydroxyurea raised some intriguing questions about mitotic control factors 26,z7,69. These arrested yeast cells contain high levels of H 1 kinase activity, which is a commonly accepted indicator of M phase. Although it could be argued that these in vitro measurements might not reflect accurately the levels of MPF in vivo, Stueland et al. 69 were able to demonstrate a molecular difference between the cdc2-cyclin-B complex from checkpoint-arrested versus untreated cells. Specifically, the presumed cdc2-cyclin-B complex from the checkpoint-arrested cells, although catalytically active, was significantly smaller during gel filtration chromatography. These experiments might suggest that either the association of additional factors with cdc2-cyclin-B complex or the state of oligomerization of this complex 7° may be important for mitotic induction by affecting its recognition of substrates, intracellular location, or other properties. Finally, the importance of other biochemical activities that may rival cdc2 in controlling mitotic events ~:annot be dismissed. In conclusion, much is known about specific steps in mitotic regulation, but a coherent picture that explains the overall control of the process has not yet emerged. It is likely that additional mitotic regulatory factors will be discovered and that there will be more surprises about the biochemistry of the well-known mitotic control enzymes. In addition, the rapid progress in the elucidation of the regulation of other cell cycle events such as the G1-S transition will provide wluable clues about G2-M regulation, and vice versa. Ultimately, a complete mechanistic under. standing of cell cycle enzymes will lead to a better appreciation of the mechanlsnls underlying carcino. genesis, terminal differentiation, and senescence. References 1 NURSE,P. (1990) Nature 344, 503-~08 2 DUNPHY,W. G., BRIZUELA,L., BEACH,D. and NEWPORT,J. (1988) Cell 54, 423-431 3 GAUTIER,j., NORBURY,C., LOHKA,M., NURSE,P. and MALLER,J, (1988) Ce//54, 433-439 4 MURRAY,A, W, and KIRSCHNER,M. W. (1989) Nature 339, 275-280 5 GLOTZER,M., MURRAY,A. W. and KIRSCHNER,M. W. (1991) Nature 349, 132-138 6 SOLOMON,M. J., HARPER,J. W and SHUTTLEWORrH,J. (1993) EMBO[ 12, 3133-3142 7 POON, R. Y. C., YAMASHITA,K., ADAMCZEWSKI,J. P., HUNT, T. and SHUI"I'LEWORTH,J. (1993) EMBO/. 12, 3123-3132 8 FESQUET,D, etaL (1993) EMBOJ.12, 3111-3121 9 RUSSELL,P. and NURSE,P. (1987) Ce1149,569-576 10 FEATHERSTONE,C. and RUSSELL,P. (1991) Nature 349, 808-811 PARKER,L. L., ATHERTON.FESSLCR,S. and PIWNICA-WORMS,H. (1992) Proc. NatlAcnd. Sol.USA89, 2917-2921 12 PARKER,L. L, and PIWNICA-WORMS0H, (1992) Science257, 1955-1957 13 McGOWAN, C. H. and RUSSELL,P. (1993) EMBOI. 12, 75-85 14 RUSSELL,P. and NURSE,P. (Iq86) Ce1145,145-153 15 UUNPHY,W. G, and KU,~:~AGAI,A. (1991) Cell67, 189-196 16 GAUTIER,]., SOLOMON, M. I., BOOHER,R. N., BAZAN,I. F. and KIRSCHNER,M. W. (1991) Ce1167,197-211
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Protein tyrosine kinases (PTKs) possess a catalytic domain capable of phosphorylating tyrosine residues on substrate proteins. The importance of this domain for the processing, via cell surface receptors, of extracellular growth-regulatory signals, together with the cacogenic potential of PTKcatalytic domains, underscores the role of tyrosine phosphorylation in the
JAK protein tyrosine kinases: their role in cytokine slgna!,l.-g
regulation of intracellular signal transduction processes. Many extraceilular signals are processed by transmembrane receptors that possess an intracellular PTK domain (Fig. la). Ligand stimulation leads to receptor oligomerization and activation of the kinase
domain, reflected by the phosphorylation of strategically located tyrosine residues in the cytoplasmic portion of the protein. The phosphotyrosines in turn act as recruitment sites for a diverse class of intracellular molecules characterized by the possession of one or more SH2 domainst.a; these domains are able to recognize and bind to phosphorylated tyrosine residues selectively. This highly specific recognition, coupled with the diverse properties of SH2.domain proteins, appears to determine the spectrum of signals transduced in response to a given stimulus. Thus the cellular response to a specific extracellular trigger can be precisely tailored to evoke the appropriate signal transduction pathways by the 'designing' of a receptor complex to include a particular constellation of SH2-domain-binding sites. Cell surface receptors of the cytokine receptor superfamily do not possess intraceilular PTK domains themselves, but many cytokines are known to induce the rapid tyrosine phosphorylation of intracellular proteins, including their own receptors. It has now become clear that these receptors compensate for their lack of an intrinsic PTK activity by recruiting and/or activating intracellular PTKs in response to ligand stimulation (Fig. lb). As is the case for the receptor PTKs, the specificity of signal transduction in response to a given signal may be determined by the constellation of molecules involved in the particular phosphotyrosine-SH2-domain interactions. Recent experiments have revealed that among the known intracellular PTKs recruited by this class of receptor are members of the JAK PTK family. TRENDS IN CELL BIOLOGY VOL. 4 JUNE 1994
•
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: A~dre~ Ziem e~kil Ail~a GI H~rpur
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Protein tyrosine kinases (PTKs) are integral components of the cellular machinely that mediates tile transduction and~orprocessing of man), extra, and intracellular signals. Members of the JAK fi,nily of intracellular PTKs (JAKI, JAK2 and TYK2) are chanlcterized by tile possession of a PTK.wlated domain and five additional homology domains, in addition to a classical PTK domain. An important breakthrough ill the understanding of lAK kinase fimction(s) has come fi'om the recent obselvations that many cytokine receptors compensate for their lack of a PTK domain by utilizing members of the JAK family fi~rsignal transduction.
The JAKfamily of PTKs Members of the JAK [an a-ronym of both Janus kinase 0anus, the Roman god of gateways, had two faces) and 'just another kinase'] family of PTKs are characterized by the possession, in addition to a bona fide kinase domain, of a kinase-related domain and five further conserved domains (Fig. 21. in contrast to almost all wholly intracellular PTKs, the members of the JAK family bear no SH2 or SH3 domains. © 1994 ElsevierScienceLid 0962.8924/94/$0700
207
f
I
The decision to enter mitosis
The phosphotyrosine content of the cdc2 protein kinase, the catalytic component o[ maturation-promoting [actor OVIPF),is an important parameter o[ mitotic regulation in a variety of organisms. Recent studies have shed considerable light on how the cdc2-specific tyroshle kinase (wee1) and its competing phosphatase (cdc25) are regulated during the cell cycle. A goal [or the Future will be to obtain a comprehensive picture of how the wee1--cdc25 regulatory system collaborates with other steps in mitotic activation to ensure that cell division occurs at the appropriate tiine during the cell cycle.
WilliamG, Dunphyis at the Divisionof Biology 216-76, California Instituteof Technology, Pasadena,CA 91125, USA,
In all eukaryotes, a variety of cyclin-dependent prorein klnases drive the Initiation and progression of each successive phase of the cell cycle t. The onset of mitosis Is coupled closely to the activation of MPF, a heterodimerlc complex that contains a B-type cyciln and the cdc2 protein kinase ~4. Except for its destruction at the metaphase-anaphase transition s, rela-
tively little Is known about post-translational regu. latlon of the cyclln subunlt of MPF. By contrast, a
OAK
great deal is known about the molecular control of cdc2 catalytic activity prior to cell division. Several cdc2-specific stimulatory and inhibitory kinases play opposing roles in the cdc2 activation process (Fig. 1). A stimulatory kinase known as cdk-activating kinase (CAK) phosphorylates Thr 161, thereby allowing cdc2 to phosphorylate its substrates 6-8. Conversely, an inhibitory kinase (weed phosphorylates cdc2 on Tyrl5, thus impeding its activity9-~. It is also thought that a distinct inhibitory kinase 12,13collaborates with weel by phosphorylating cdc2 on an adjacent residue (Thrl4). One of the final events in the initiation of mitosis is the tyrosine dephosphorylation of cdc2 by the cdc25 protein, an extremely specific and highly regulated phosphatase 1.-)7. In fission yeast, competition between the cdc25 and weel proteins appears to be a critical determinant of mitotic timing t. For this reason, there has been considerable interest in how the actions of these proteins are regulated during the cell cycle. Recent studies indicate that the catalytic activities of both the cdc25 and weel proteins are controlled tightly by an upstream network of kinases and phosphatases ls-z2. However, it is not entirely clear how these phosphorylation reactions, which ultimately regulate the phosphotyrosine content of cdc2, contribute to the initial decision to enter mitosis. Another issue is that the relative importance of tyrosine phosphorylation as a cdc2-regulatory mechanism appears to vary considerably depending upon the particular characteristics of the cell cycle tn a given organism z3.z7. This apparent lack of universality raises an important question: does the underlying mechanism for controlling the Initiation of mitosis differ among species or Is the basic mechanism conserved, with refinements to accommodate the needs of particular species? The widespread interest In this question has been heightened by the likely possibility that the mitotic decision mechanism is controlled by cell cycle checkpoint factors, the proreins that render the Initiation of mitosis dependent upon the successful completion of earlier cell cycle processes such as accurate DNA replication 2~,z9,
cdo25
[ .o,,n
1
1" l l-
v
+
wee1
G2
Mitosis
niml FIGURE 1 Post-translational regulation of the cdc2-cyclin.B complex. Cdk-activating kinase (CAK) stimulates cdc2 activity by phosphorylating Thr161. The cdc25, wee1 and niml proteins cont,ol the inhibitory tyrosine phosphorylation of cdc2 on Tyrl S. 202
© 1994ElsevierScienceLtd 0962-89241941507.00
Regulation of the cdc2S protein by phosphorylatlon reactions It is well established that the cdc25 protein is weakly active during interphase and becomes fully active at the G2-M transition owing to an extensive phosphorylation of its N-terminal regulatory domain at approximately six sites 19,2".a~z. During interphase, the cdc2S.stimulatory kinase is weakly active, and a potent cdc25.inhibitory phosphatase opposes its action (Fig. 2). This inhibitory phosphatase is quite sensitive to okadaic acid and has several characteristics reminiscent of protein phosphatase 2A (PP2A). Both the cdc25-regulatory enzymes are tightly regulated in a manner consistent with their role in controlling the activation of the cdc25 protein: at the G2-M transition, the activities of the cdc25-stimulatory kinase and cdc25-inhibitory phosphatase increase and decrease, respectively ~9. The identity of the cdc25-stimulatow kinase has been examined in a number of studies, cdc2--cyclin-B TRENDSIN CELLBIOLOGYVOL, 4 JUNE1994
i can phosphorylate cdc25 well/n vitro3~.3z. However, Xenopus extracts containing little or no cyclin B can effect the phosphorylation of cdc25 efficiently once PP2A action is blocked by okadaic acid ~9, The recent studies of Kuang et al. 3~may help to clarify this issue. They demonstrated that the cdc25 protein is a substrate of the ME (MPM-2 epitope) kinase, the enzyme responsible i¢,r the creation of a phosphopeptide epitope in a myriad of mitotic phosphoproteins recognized by the MPM-2 monoclonal antibody. One possibility is that the ME kinase, cdc2 and perhaps other kinases all contribute to the activation of cdc25. In this regard, an important issue is whether the ME kinase is regulated by cdc2 or is controlled independently. In the former case, the ME kinase would serve as an intermediary in the cdc2-dependent, self-perpetuating activation of cdc25, whereas in the latter case the activation of the cdc25 protein could be modulated by events that precede the switching-on of the cdc2 protein. This latter possibility is especially intriguing in view of the fact that biochemical activities in Xelzopus egg extracts distinct from cdc2-cyclin-B appear to be able to act as MPF under certain circumstances 34-:~6.
Regulation of the wee1 protein by multiple klnases Not surprisingly, in vitro experiments have suggested the activity of the weel proteln is highly regulated during the cell cycle. The first clue about wee1 regulation was provided by genetic experiments that identified the niml protein kinase as a negative regulator of the weel pathway in fission yeast:~7,:~8.These studies were validated by the demonstration that recombinant niml protein can phosphorylate and completely inactivate the isolated wee] protein in a cell-free reaction :¢~4=. A notable feature of the nlmlcatalysed phosphowlation of the weel protein Is that It is restricted to the C.terrnlnal region of the protein that forms its kinase domain, suggesting a potentially direct effect on catalytic function, in fission yeast, the action of the niml protein is involved in the nutritional regulation of mitotic timing :.7,:~. At present, a niml homologue from higher somatic cells has not been described, and Xe,opus egg extracts appear to lack a niml-like activity, as judged by biochemical assays. For these reasons, it remains to be established whether phosphorylation of the catalytic domain of the weel protein by niml-like kinases is a widely used mechanism for mitotic regulation in higher eukaryotic cells where physiological parameters other than nutrient availability are more crucial. Although the niml protein is clearly able to modulate the activity of the weel protein over a wide range, a variety of arguments suggested that it would not be the only regulator of weel. Indeed, mitotic Xenopus egg extracts were shown to contain a kinase activity specific for the N-terminal region of recombinant fission yeast weel, which effected an -60 kDa increase in the apparent molecular mass of weel on SDS-gei electrophoresis 4z. This extensive phosphoryiation, like the more modest pho.~phorylation catalysed by the niml protein, leads to the almost complete inactivation of the ability of weel to
TRENDSIN CELLBIOLO~3YVOL. 4 IUNE 1994
cdc25-stimulatory kinase(s)
~
>
~
cdc25 PP2A
mitosis
interphase
FIGURE2 Regulation of cdc25 activity.The phosphatase activityof the cdc25 protein is modulated by both a cdc25-stimulatory kinase that phosphorylates its N-terminal, regulatory domain and a cdc25-inhibitory phosphatase (shown here as PP2A)that counteracts the activation process. phosphorylate cdc2. The identity of the weelinhibitory kinase(s) in these in vitro experiments has not been established with certainty, but cdc2depleted extracts maintained in M phase with phos. phatase inhibitors contain high levels of the activity. The inhibitory phosphorylation of the N-terminal region of the weel protein is counteracted by an okadaic-acid-sensitive phosphatase (PP2A-like) that is highly active in interphase Xe,opus egg extracts and thus maintains weeI in its active form. Since it is not yet established whether somatic cells such as fission yeast contain the N-terminal-specific, weelinhibitory kinase, it is not known how this activity and the him 1 protein kinase might collaborate with one another in weel regulation (Fig. 3).
Coordination of cdc25 and wee1 regulation There are some striking parallels between the regulation of cdc25 and weel. Both proteins are
low activity
high activity
weel-lnhibitory klnase(s)
wee1
.,= PP2A
n''lT?
Y
low activity FIGURE3 Two kinase-phosphatasesystemsregulatewee1 activity. The niml protein and a competing phosphatasecontrol the phosphory!ationof the catalyticdomain of the wee1 protein. In addition, a distinct kinaseactivity can shutoff wee1 by phosphorylatingits N-terminaldomain, and is counteractedby a PP2A.qke phosphatase. 203
III III I I II
I I
I
NI
Ic
cdc2 does not seem to serve as a potent catalyst for its own activation 46,47. It seems that there is more of the mitotic puzzle to be deciphered.
cdc25
I I
111 II III I
I IIIII
II
"1
•
II
Ic
wee1 FIGURE 4 Potential phosphorylation sites in the XenopusC-type cdc25 protein (see Ref. 19) and the fission yeast wee1 protein (~ee Ref. 9). The location of Ser/Thr-Pro motifs (14 in total for cdc25 and 25 in total for wee1 ) are indicated with a bar. In addition, the locations of catalytically critical residues (Cys for cdc2S and Lys for wee1) are shown.
extensively phosphorylated at the G2-M transition on N-terminal regulatory domains that contain a high density of Ser/Thr-Pro motifs that are potential substrates for a variety of mitotic kinases (Fig. 4). The mechanism by which this phosphorylation influences the function of either the cdc25 or weel protein is not known, in the case of the cdc25 protein, the phosphorylation could either enhance the recognition of the cdc2-cyclin-B complex or relieve a potentially inhibitory effect of the N-terminal domain on the Cterminal catalytic region. Conversely, phosphorylation of the weel protein might either hinder the recognition of the cdc2--cyclin.B complex or result in the inhibition of the C-terminal kinase domain. In addition, the possibility that the phosphorylation of the cdc25 and wee1 proteins might have additional effects on other processes such as intracellular localIzation cannot be excluded. For both the cdc25 and weel proteins, the upstream klnase=phosphatase system Is balanced such that during Interphase both cdc2S and weel remain In an underphosphorylated state, corre. spondlng to their low. and hlgh-actlvlty versions, respectively. A PP2A-Ilke phosphatase has been implicated in both cdc25 and weel regulation, rals. Ing the possibility that the same enzyme might inhibit cdc25 and stimulate weel during inter. phase tg,3°,42-4s. There is evidence that the cdc25inhibitory, PP2A-like phosphatase is downregulated at mitosis tg. Similarly, total kinase activity towards both cdc2S and weel Is greatly elevated at mitosis, the period when cdc2 is most active. Such phosphorylation.-dephosphorylatlon circuits would appear to guarantee the rapid and synchronous (i,e. self-perpetuating) activation of cdc2 at the G2-M transition. However, this scheme suggests a paradox in that the activation of cdc2 requires prior activation of cdc25 and/or inactivation of weel, which in turn would be dependent upon active cdc2. The question arises as to what molecular event tips the balance to favour production of the active cdc2 that would set this activation process in motion. In principle, any factor that could stimulate cdc2S or inhibit weel might be a candidate. However, this triggering event need not involve tyrosine phosphorylation directly. Another consideration is that a low level of active 204
Intracellular localization of cdc2 and its regulators at the G 2 - M transition
The role of intracellular localization in cdc2 regulation is attracting increasing interest. The entry of cdc2-cyclin-B into the nucleus near the time of prophase is well established4s-s°, but the precise role of nuclear translocation in controlling both the actiration and action of the cdc2 protein remains to be determined. The regulators of cdc2 (e.g. cdc25 and weel) likewise display suggestive intracellular locations during the cell cycle. Most investigators appear to concur that the cdc25 protein is largely nuclear in the period just before mitosissl-s4. However, during the remainder of interphase, the cdc2S protein has been observed in either the cytoplasm or the nucleus depending upon the experimental system. For this reason, it is unclear whether the regulated entry of cdc25 into the nucleus is a general feature of mitotic control. Recently, studies on the intracellular location of weel have provided a potentially important perspective on cell cycle regulation. Heald et al. s4 transfected baby hamster kidney (BHK) cells with a gene encoding the human weel-like tyrosine kinase and assessed both the intracellular localization of the expressed protein and its effect on the activity of cdc2-cyclin.B (whose expression was also directed by transfected plasmids). The transfected weel protein was ahnost exclusively nuclear, and the cdc2-cyclinB complex in tile nucleus was apparently inactive, as evidenced by the absence of nuclear mitotic events such as chromosome condensation. By contrast, cytosolic cdc2-cyclin-B remained active as Judged by its ability to induce the depolymerization of a recombinant lamln protein containing a mutation that forced it to remain outside the nucleus. The interesting conclusion posed by these studies is that the activity of the cdt.2-cyclln-B complex may be regulated at least in part by its location in the cell, thus adding a layer of complexity to mitotic regulatory mechanisms. These studies also raise some interesting questions. It might be expected that cytosollc cdc2-cyclln-B is normally under some form of regulation. Is there a distinct cytosollc weel protein or do other regulatory mechanisms not involving tyroslne phosphorylation predominate in the cytoplasm? Role of cdc25 and wee1 proteins In cell cycle checkpoints
in 1989, Hartwell and Welnert introduced the concept of checkpoints to cell cycle control 2x. A checkpoint refers to the process whereby a cell can undergo an arrest or delay of cell cycle progression in response to the perturbation of a cellular process that might ultimately interfere with the successful production of two viable daughter cells, Well-known checkpoints include the block to mitosis in the presence of replicating DNA, the arrest of the cell cycle at any one of several points after DNA damage, and the delay of anaphase in response to the improper association of TRENDS IN CELL BIOLOGY VOL. 4 JUNE 1994
I the chromosomes with the mitotic spindle (see Ref. 29 for a review of checkpoints). In the case of mitotic control, the mechanisms underlying the checkpoint that imposes a G2-1ike arrest of the cell cycle in the presence of unreplicated or damaged DNA have been studied in several experimental systems. In fission yeast, a growing number of genes (e.g. tad1, rad3, radO, rad17 and husl) have been implicated in this process ss-s7. A similar class of genes has been defined in budding yeastsS. An important question is how these gene products impinge upon the activation and/or action of MPF at the G2-M transition. In fission yeast, the cdc2S and weel proteins are clearly involved in a checkpoint-like mechanism that controls the size at which cell division occurs. Consequently, the question arose as to whether the cdc2-regulatory tyrosine kinase-phosphatase system participates in other checkpoints such as those involving unreplicated and/or damaged DNA. The observation that strong overexpression of the cdc25 protein in fission yeast disrupted the replication checkpoint was broadly consistent with this notion so. However, the overexpression of cdc25 did not abolish the DNA-damage checkpoint, suggesting that the replication and damage checkpoints utilize different mechanistic pathways. Moreover, further analysis6° indicated that although cdc25 can influence the replication checkpoint under certain circumstances it appears not to be the pivotal enzyme in the process: for example, a certain weel;cdc25 double mutant strain that is viabl ~. without producing the cdc25 protein can arrest normally in the presence of a DNA synthesis inhibitor (e.g. hydroxyurea). Similarly, the steady-state activity of the cdc25 protein in lnterphase Xcnopus egg extracts is unaffected by the presence of unrepllcated DNA ~'~,2°. The role of the weel protein in the replication checkpoint has also been examined in detail. Fission yeast mutants lacking the activity of either weel or mikl (a related klnase that appears to have a similar function to weel) reportedly both delay mitosis In the presence of hydroxyurea, but a double mutant lacking both weel and mikl activities is profoundly deficient in the replication checkpoint ~:4.sT.so. This observation is consistent with the fact that a fission yeast strain harbouring a mutant version of cdc2 that cannot undergo tyrosine phosphorylation on residue 1S is also deficient in the replication checkpoint 6°. Moreover, experiments in Xenopus egg extracts revealed that the inhibition of DNA replication leads to an elevation of total tyrosine kinase activity specific for cdc22~.6L Nonetheless, in both the fission yeast and frog systems, it has not yet been demonstrated whether the regulation of tyrosine kinase action specific for cdc2 is a direct consequence of the monitoring of unreplicated or replicating DNA. This information will aid understanding of cell cycles in other systems where the tyrosine phosphorylation of cdc2 appears to be less critical. Mitotic regulation without phosphotyroslne
There is now ample precedent for proper mitotic regulation in the absence of apparent tyrosine TRENDSIN CELLBIOLOGYVOL. 4 JUNE1994
phosphorylation on the cdc2 protein. Perhaps the first example described was the early Xenopus embryo, where tyrosine phosphorylation of cdc2 is not detectable during cleavages 2-12 fret. 62). However, the definitive demonstration was provided in the budding yeast where it was shown that strains harbouring an altered cdc2 (CDC28) protein that could not be phosphorylated on the equivalent of Tyrl5 (as well as the preceding threonine residue) could nonetheless grow and divide normally26.27. Strikingly, the replication and repair checkpoints were also fully operational in these mutant strains. These observations have been validated by genetic analysis of the budding yeast weel homologue called SWE163: budding yeast lacking the SWE1 protein divide normally and still can delay mitosis in response to DNA damage. In this regard, it is intriguing that budding yeast do contain an operative weel--cdc2S system (i.e. the SWE1 protein and the cdc25 homologue MIH1; see Ref. 64). One notion is that the function of this system has become iargely cryptic in an organism such as budding yeast that does not rely on a G2 size control in its budding mode of division. Alternatively, the budding yeast weel--cdc25 system may be keyed into physiological parameters that are not readily apparent. An important question is whether mitotic control systems that can operate properly without tyrosine phosphorylation of cdc2 represent special cases or instead reveal a more universal mode of G2-M regulation.
Other mechanismsfor mitotic regulation Although this article has focused heavily on the cdc25 and weel proteins, there are other factors that contribute to mitotic regulation. For exalnple, the phosphorylation of cdc2 on Thr161 is an important prerequisite for its catalytic activity. The catalytic subunit of CAK has recently been identified as the Me15 klnasec'~s. These studies presented data that CAK may possess a partner analogous to a cyciln, and it Is possible that CAK itself is also regulated by phosphorylatlon. In view of this potential complexity, the regulation of Thrl61 phosphorylation by CAK or its competing phosphatase would be a logical target for mitotic control mechanisms, but the evidence to date suggests that the level of Thrl61 phosphorylation is constitutive dmmg the cell cycle6s. During the G1 phase of the cell cycle, a number of small inhibitor proteins, the cyclin kinase inhibitors (CKIs), can bind to and inhibit the catalytic activity of various G l-specific cdks such as cdk2-cyclin-E (see Refs 66 and 67 for reviews). Analogous proteins have not been reported to interact with cdc2 during G2, but the existence of such factors could resolve some of the apparent paradoxes about G2-M regulation. Parenthetically, it is well known that the fission yeast sucl protein (13 kDa) and homologous proteins in other species bind efficiently to cdc2, but these polypeptides appear not to inhibit the catalytic activity" of cdc2, and there is no clear agreement about their involvement in other steps of cdc2 biochemistry~'~. The studies of cdc2--cyclin-B regulation in budding yeast arrested at the replication checkpoint by 20S
treatment with hydroxyurea raised some intriguing questions about mitotic control factors 26,z7,69. These arrested yeast cells contain high levels of H 1 kinase activity, which is a commonly accepted indicator of M phase. Although it could be argued that these in vitro measurements might not reflect accurately the levels of MPF in vivo, Stueland et al. 69 were able to demonstrate a molecular difference between the cdc2-cyclin-B complex from checkpoint-arrested versus untreated cells. Specifically, the presumed cdc2-cyclin-B complex from the checkpoint-arrested cells, although catalytically active, was significantly smaller during gel filtration chromatography. These experiments might suggest that either the association of additional factors with cdc2-cyclin-B complex or the state of oligomerization of this complex 7° may be important for mitotic induction by affecting its recognition of substrates, intracellular location, or other properties. Finally, the importance of other biochemical activities that may rival cdc2 in controlling mitotic events ~:annot be dismissed. In conclusion, much is known about specific steps in mitotic regulation, but a coherent picture that explains the overall control of the process has not yet emerged. It is likely that additional mitotic regulatory factors will be discovered and that there will be more surprises about the biochemistry of the well-known mitotic control enzymes. In addition, the rapid progress in the elucidation of the regulation of other cell cycle events such as the G1-S transition will provide wluable clues about G2-M regulation, and vice versa. Ultimately, a complete mechanistic under. standing of cell cycle enzymes will lead to a better appreciation of the mechanlsnls underlying carcino. genesis, terminal differentiation, and senescence. References 1 NURSE,P. (1990) Nature 344, 503-~08 2 DUNPHY,W. G., BRIZUELA,L., BEACH,D. and NEWPORT,J. (1988) Cell 54, 423-431 3 GAUTIER,j., NORBURY,C., LOHKA,M., NURSE,P. and MALLER,J, (1988) Ce//54, 433-439 4 MURRAY,A, W, and KIRSCHNER,M. W. (1989) Nature 339, 275-280 5 GLOTZER,M., MURRAY,A. W. and KIRSCHNER,M. W. (1991) Nature 349, 132-138 6 SOLOMON,M. J., HARPER,J. W and SHUTTLEWORrH,J. (1993) EMBO[ 12, 3133-3142 7 POON, R. Y. C., YAMASHITA,K., ADAMCZEWSKI,J. P., HUNT, T. and SHUI"I'LEWORTH,J. (1993) EMBO/. 12, 3123-3132 8 FESQUET,D, etaL (1993) EMBOJ.12, 3111-3121 9 RUSSELL,P. and NURSE,P. (1987) Ce1149,569-576 10 FEATHERSTONE,C. and RUSSELL,P. (1991) Nature 349, 808-811 PARKER,L. L., ATHERTON.FESSLCR,S. and PIWNICA-WORMS,H. (1992) Proc. NatlAcnd. Sol.USA89, 2917-2921 12 PARKER,L. L, and PIWNICA-WORMS0H, (1992) Science257, 1955-1957 13 McGOWAN, C. H. and RUSSELL,P. (1993) EMBOI. 12, 75-85 14 RUSSELL,P. and NURSE,P. (Iq86) Ce1145,145-153 15 UUNPHY,W. G, and KU,~:~AGAI,A. (1991) Cell67, 189-196 16 GAUTIER,]., SOLOMON, M. I., BOOHER,R. N., BAZAN,I. F. and KIRSCHNER,M. W. (1991) Ce1167,197-211
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rewe~,s
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Protein tyrosine kinases (PTKs) possess a catalytic domain capable of phosphorylating tyrosine residues on substrate proteins. The importance of this domain for the processing, via cell surface receptors, of extracellular growth-regulatory signals, together with the cacogenic potential of PTKcatalytic domains, underscores the role of tyrosine phosphorylation in the
JAK protein tyrosine kinases: their role in cytokine slgna!,l.-g
regulation of intracellular signal transduction processes. Many extraceilular signals are processed by transmembrane receptors that possess an intracellular PTK domain (Fig. la). Ligand stimulation leads to receptor oligomerization and activation of the kinase
domain, reflected by the phosphorylation of strategically located tyrosine residues in the cytoplasmic portion of the protein. The phosphotyrosines in turn act as recruitment sites for a diverse class of intracellular molecules characterized by the possession of one or more SH2 domainst.a; these domains are able to recognize and bind to phosphorylated tyrosine residues selectively. This highly specific recognition, coupled with the diverse properties of SH2.domain proteins, appears to determine the spectrum of signals transduced in response to a given stimulus. Thus the cellular response to a specific extracellular trigger can be precisely tailored to evoke the appropriate signal transduction pathways by the 'designing' of a receptor complex to include a particular constellation of SH2-domain-binding sites. Cell surface receptors of the cytokine receptor superfamily do not possess intraceilular PTK domains themselves, but many cytokines are known to induce the rapid tyrosine phosphorylation of intracellular proteins, including their own receptors. It has now become clear that these receptors compensate for their lack of an intrinsic PTK activity by recruiting and/or activating intracellular PTKs in response to ligand stimulation (Fig. lb). As is the case for the receptor PTKs, the specificity of signal transduction in response to a given signal may be determined by the constellation of molecules involved in the particular phosphotyrosine-SH2-domain interactions. Recent experiments have revealed that among the known intracellular PTKs recruited by this class of receptor are members of the JAK PTK family. TRENDS IN CELL BIOLOGY VOL. 4 JUNE 1994
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Protein tyrosine kinases (PTKs) are integral components of the cellular machinely that mediates tile transduction and~orprocessing of man), extra, and intracellular signals. Members of the JAK fi,nily of intracellular PTKs (JAKI, JAK2 and TYK2) are chanlcterized by tile possession of a PTK.wlated domain and five additional homology domains, in addition to a classical PTK domain. An important breakthrough ill the understanding of lAK kinase fimction(s) has come fi'om the recent obselvations that many cytokine receptors compensate for their lack of a PTK domain by utilizing members of the JAK family fi~rsignal transduction.
The JAKfamily of PTKs Members of the JAK [an a-ronym of both Janus kinase 0anus, the Roman god of gateways, had two faces) and 'just another kinase'] family of PTKs are characterized by the possession, in addition to a bona fide kinase domain, of a kinase-related domain and five further conserved domains (Fig. 21. in contrast to almost all wholly intracellular PTKs, the members of the JAK family bear no SH2 or SH3 domains. © 1994 ElsevierScienceLid 0962.8924/94/$0700
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