Protein

phosphatases

Catherine

J. Pallen, Y.H. Tan and Graeme

Institute

of Molecular

in cell signalling

and Cell Biology, National University Singapore, Republic of Singapore

Protein phosphorylation is the most common ion and plays a role in all known pathways phosphorylation is a result of the balance of and phosphatases. There is increasing evidence of phosphate groups from proteins, catalyzed required for the downstream activation of

Current

Opinion

in Cell Biology

Introduction This review summarizes some recent advances that pinpoint both serine/threonine and tyrosine phosphatases as key participants in signalling pathways. Phosphatases can be directly or indirectly involved; either a dephosphorylation event is required to activate downstream signalling components, or the restraint of dephosphorylating activity is necessary to permit phosphorylation and signal propagation. In some cases the relevant phosphatase has been positively identified, while in other cases the involvement of a phosphatase has only been implied. We have emphasized the regulation of phosphatase activity where it has been identified; diverse regulatory mechanisms include phosphorylation/dephosphorylation, protein-protein interaction with activators or inhibitors and regulation by second messengers.

The protein

phosphatases

Protein phosphatases are classified as serine/threonine phosphatases or as tyrosine phosphatases according to amino acid sequence and substrate selectivity. There are four major classes of serine/threonine phosphatases (PP-1, PP-2A, PP-2B, PP-2C), each of which has isozymic forms, and several related but distinct novel enzymes (reviewed in [ 11). The tyrosine phosphatases can be classed as either receptor-like or non-receptor molecules, and they encompass a rapidly expanding number of members (reviewed in [ 21).

R. Guy of Singapore,

post-translational modificatof signal transduction. Net activities of protein kinases that the regulated removal by protein phosphatases, is other signalling proteins.

1992, 4:100&1007

Phosphatases transduction Phosphatase

in membrane-to-nuclear

inhibition

Very few serine/threonine phosphatases, with the recent exception of calcineurin (see below), have been identified as active participants in signal transduction pathways, although they are certainly involved in signal attenuation. This is supported by the finding that specific inhibitors of protein phosphatases-1 and -2A (PP-1, PP-2A), such as okadaic acid, calyculin A, tautomycin and microcystin, rarely inhibit, but often mimic, various aspects of signal transduction, including enhanced phosphorylation and activation of signalling enzymes and activation of transcription factors and genes. In most of these studies the inhibited phosphatase in a particular pathway has not been located, and it is difficult to distinguish whether inhibition mimics a physiological phosphatase inhibition or merely reproduces the same effect as physiological activation of a kinase, or both. The ligandinduced inhibition of phosphatases in signal transduction is not a well explored area of research. The use of phosphatase inhibitors in suggesting such a possibility has been demonstrated by the initial finding that okadaic acid closely mimics aspects of tumour necrosis factor (TNF)/interleukin (IL)-1 signalling [?I], and the subsequent identification of a phosphatase activity that is rapidly repressed following treatment of human fibroblasts with TNF/IL-1 [4**]. This may also be found to be the case in other pathways that are mimicked by okadaic acid.

Abbreviations CSA-cyclosporin

1000

A; CYP-cyclophilin; FKBP-FK-binding protein; IL-interleukin; PMA-phorbol 12-myristate lbacetate; PP-protein phosphatase; TCR-T-cell receptor; TNF-tumor necrosis

@ Current

Biology

NFAT-nuclear rdgC-retinal factor.

Ltd ISSN 0995-0674

factor of activated degeneration C;

T cells;

Protein Drosophila

phosphatases

A tyrosine phosphatase encoded by the gene corkscrew participates in a signal-transduction pathway that determines terminal cell fate in Drosophila [5**]. The corkscrew phosphatase acts distal to a receptor tyrosine kinase, torso, and in conjunction with the Raf serine/threonine kinase it effects the downstream activation of two genes (hucklebein and taz2Ies.s) that encode putative transcription factors. The corkscrew phosphatase is a non-receptor type enzyme containing two amino-terminal src homology (SH) 2 domains, suggesting that Corkscrew protein function or regulation involves the recognition and binding of phosphotyrosyl-containing proteins. Several similar mammalian tyrosine phosphatases with SH2 domains have been cloned [6*-8*] and could also be involved in transducing signals from receptor tyrosine kinases. A novel serine/threonine protein phosphatase encoded by the Drosophila retinal degeneration C (rdgC) gene is required to prevent light-induced retinal degeneration [9**]. The RdgC protein has approximately 30 % homology with the catalytic domains of PP-1, PP-2A and PP-2B, and the carboxyl-terminal half contains five potential Ca*+-binding sites, suggesting that phosphatase activity is regulated by Ca*+. The photoreceptors of flies with an rdgC mutation degenerate if the flies are reared in light. This effect is mediated by light-activated rhodopsin, but does not require activation of the norp A encoded phospholipase C, a downstream component of the rhodopsinG protein phototransduction cascade. The rdgC phosphatase may participate in an alternative rhodopsin-initiated pathway that regulates photoreceptor membrane renewal [lo].

Phosphatases

in T-cell

activation

At least two protein phosphatases are essential components of the T-cell receptor (TCR) signal transduction pathway. The receptor-like tyrosine phosphatase, CD45, is involved in early membrane-associated events, and the serine/threonine phosphatase calcineurin (PP-2B) mediates a subsequent downstream step. Stimulated T cells lacking CD45 are unable to generate the phosphatidylinositol-derived second messengers inositol trisphosphate and Ca*+ [ 111, probably due to their failure to activate tyrosine kinases [12**] that couple the TCR to the phosphatidylinositol pathway (reviewed in [ 13151). Candidate coupling kinases and CD45 substrates include the CD4associated Lck kinase and the TCR 6 chain associated Fyn kinase, both of which are proposed to be activated by dephosphorylation of a regulatory carboxyl-terminal tyrosine residue. The mechanism regulating CD45 activity in response to antigen binding is unknown, but activation could involve the association of CD45 with the TCR, CD4 or other Tcell surface proteins. Decreased CD45 activity and a loss of serine phosphate from CD45 occur in response to an ionomycin-induced increase in intracellular Ca* + [ 16**], suggesting that a negative feedback loop comprising the

phosphatases

in cell signalling

Pallen, Tan, Guy

Ca*+ stimulated dephosphorylation of CD45, possibly by calcineurin, could restore CD45 activity to resting levels. The immunosuppressant drugs cyclosporin A (CM) or FK506 bind to intracellular receptors called immunophilins [ cyclophilin (CyP) or FK-binding protein (FKBP), respectively] and interfere with a downstream signaIling step in T-cell activation. Both drugs effect a remarkably similar and selective inhibition of the Ca*+-sensitive transcription of a subset of early phase T-cell activation genes, including the gene that encodes IL-2 (reviewed in [ 171). The drug-immunophilin complexes of CsACyP and FK506-FKBP (but not CyP or FKBP alone) were recently shown to bind to and inhibit the activity of the Ca*+/calmodulin-dependent phosphatase calcineurin ([18**,19”,20*]; (reviewed in [21,22]). CsA and FK506 also inhibit the activation of nuclear factor of activated T cells (NFAT), a transcription factor that regulates the Z-2 gene. They interfere with the Ca*+ -dependent nuclear transkxation of a cytoplasmic component of NFAT, which normally complexes in the nucleus with a phorbol myristate acetate (PMA)-inducible nuclear subunit to constitute active NFAT [ 23’1. Overexpression of the catalytic subunit of calcineurin stimulates NFATdependent transcription [24”,25*m], indicating that the phosphatase is coupled with and functions at a point preceeding activation of this factor. A dephosphorylation went must be a direct or indirect prerequisite for translocation. Calcineurin could dephosphoxylate NFAT itself, or act on an upstream activator of NFAT or on a substrate that inhibits or is part of the translocation machinery. Whichever the case, calcineurin has now been recognized as a component of the so-called ‘black box’ in T-cell signalling, coupling cell membrane associated and nuclear events. What is the function of calcineurin in other cell types? This phosphatase may also be involved in the CsA- and FK506sensitive irnmunoglobulin E receptor signalling pathway leading to mast cell degranulation 1261. Simultaneous mutation of two yeast calcineurin genes blocks the recovery of yeast cells from mating pheromone-induced cell-cycle arrest, suggesting that calcineurin mediates an essential Gt-S phase transition step in the mating response [ 27**]. Calcineurin and FKBP are present at high levels and co-localize in brain [ 281; both proteins, as well as CyP, are found in a variety of other tissues. Do natural ligands of the immunophilins exist that promote complexation with calcineurin and inhibition of phosphatase activity? This would be desirable if calcineurin phosphatase activity was not required in response to elevated intracellular Ca*+ levels, or if inhibition of calcineurin favored net phosphotylation and activation of signalling components in other pathways.

Unidentified

tyrosine

phosphatases

Treatment of CV-1 cells with isoproterenol, forskolin, CAMP analogues or phorbol ester results in tyrosine phosphatase activation, suggesting that the second messenger-stimulated protein kinases Aand C regulate ty-

1001

1002

Post-transcriptional

processes

rosine phosphatase activity 1291. The peptide hormone somatostatin stimulates tyrosine phosphatase activity in pancreatic cancer cells through a G-protein-mediated mechanism [30*]. These observations suggest that ty rosine phosphatases actively participate in transduction pathways, an area that warrants further investigation.

Regulation of transcription phosphatases

factor

activity

by

Phosphorylation is the primary mode of regulating transcription factor localization, DNA binding and interaction with the transcription machinery (reviewed in [31,32]). In a few instances, the ability of transcription factors to activate target genes is impaired by phosphorylation, and dephosphorylation is required for activation. The calcineurin-dependent translocation of the transcription factor NFAT has been discussed above. Nuclear translocation of another transcription factor, the yeast !WI5 protein, is effected by dephosphorylation [33*]. During the S, G2 and M phases of the cell cycle, SWI5 is retained in the cytoplasm by the CDC28 (equivalent to Cdc2)mediated phosphorylation of three serine residues close to or within the nuclear localization signal. At the anaphasffit transition these sites are dephosphotylated by an unidentified phosphatase and SW15 enters the nucleus. The inactivation of the CDC28 kinase at the end of mitosis is important for SW15 dephosphotylation. Whether an unaltered level of WI5 phosphatase activity is then sufficient to effect net dephosphotylation or whether this phosphatase undergoes a coincident activation at the end of mitosis is not known. The best example of DNA binding of a transcription factor being controlled by probable phosphatase regulation is c-Jun. Constitutive phosphotylation of three serine/threonine residues near the DNA-binding domain of c-Jun inhibits DNA binding. Exposure of cells to phorbol ester (PMA), which activates protein kinase C, induces dephosphorylation of these sites and DNA binding [34*]. There is at present no positive identification of the phosphatase whose activation is an endpoint of a protein kinase C-induced pathway, but recent evidence indicates that a protein phosphatase of unknown nature is regulated, not only by PMA and growth factors, but also by some transforming oncogenes [31 I.

Phosphatases

in cell-cycle

regulation

The activity of a key cell cycle regulatory element, the Cdc2 serine/threonine kinase or p34cdc2, is determined by its phosphorylation state (see Fig. 1). Activation of p34cdc2 (complexed with cyclin B) at the G2-M phase transition requires dephosphotylation of Thr14 and ‘Iyrl5 and continued phosphorylation at Thrlbl. Activation of a tyrosine phosphatase encoded by the c&25 gene results in dephosphotylation of Tyrl5, and probably of Thr14, in p34cdc2, and the consequent initiation

of mitosis. PP-1 and PP-2A act in pathways regulating the entry to and exit from mitosis.

Cdc25

Cdc25 was not initially recognized as a tyrosine phosphatase due to its low sequence homology with known members of the tyrosine phosphatase family, although several observations closely linked Cdc25 action to Cdc2 dephosphotylation (reviewed in [ 351). Isolated Cdc25 protein was finally shown to have intrinsic tyrosine phosphatase activity and properties characteristic of other tyrosine phosphatases, including the hallmark loss of activity upon mutation of an essential cysteine residue in the active site [36*-41*]. Two notable features of Cdc25 are its high specificity for p34cdc2 and its probable dual specificity for both Thr14 and Tyr15 in p34cdc2. Cdc25 is most homologous to the vaccinia virus tyrosine phosphatase VHl, and unlike other tyrosine phosphatases, Cdc25 and WI1 can dephosphorylate both tyrosine and serine residues in vitro [40*,42*]. In Xenopus, the activity of Cdc25 is regulated by phosphorylation/dephosphotylation and through proteinprotein interaction. Interphase Cdc25 is a phosphosetyl protein with basal phosphatase activity. Near the onset of mitosis, multiple serine and threonine residues in the amino-terminal region of Cdc25 are phosphorylated, resulting in a five-fold increase in the phosphatase activity of Cdc25 [43**]. The preponderance of serine/threonine-proline motifs in this region of Cdc25 suggests that the kinase responsible for this may be Cdc2 or a CdQ-activated kinase. Dephosphotylation of Cdc25 by an okadaic acid-sensitive phosphatase restores Cdc25 activity to interphase levels. The activities of the Cdc25-speciiic kinase and phosphatase are cell cycle-regulated, with a coincident rise in the stimulatory kinase activity and decrease in the inhibitory phosphatase activity just before mitosis [43**]. The phosphatase activity of Cdc25 is also stimulated four-fold to five-fold in vitro by B-type cyclins, which contain a potential transactivation domain homologous to a region in other tyrosine phosphatases but lacking in Cdc25 [44**]. Cdc25 undergoes a cell cycle-dependent association with the Cdc2-cyclin B complex. This association peaks at mitosis and closely preceeds maximal kinase activity of Cdc2 [ 45**]. The events described above may be features of an interlocking, positive feedback mechanism that regulates Cdc25 activity. Association of Cdc2-cyclin B with Cdc25 could sufficiently stimulate the basal activity of a portion of Cdc25 to dephosphotylate a few, or even more, molecules of Cdc2. This activated Cdc2 could directly or indirectly phosphotylate the complexed Cdc25 to bring about further activation of Cdc25, and so on. Such events would be favoured if they occurred in conjunction with a reduction in activity of the CdQ-specific tyrosine kinase (wee 1 or mik 1) and increased inhibition of the Cdc25specific serine/threonine phosphatase (see below). A cyclical and rapid accumulation of dephosphoCdc2 and phospho-Cdc25 would drive the initiation of mitosis.

Protein

phosphatases

y-----w,\

in cell

signalling

Pallen,

Tan,

Guy

MITOSIS

degredation

\

mik 1 wee 1 :inases -

1

PP-ZA?

i-7

1 PP-2A?

1

INTERPHP

Fig. 1. The known

Protein

roles of protein phosphatases as p34cdcz. See text for details.

phosphatase-I

and

-2A

in the

in the

cell

cell

cycle

cycle.

P, inorganic

PP-1 product in fission yeast acts in the pathway that regulates initiation of mitosis, but does not appear to affect Cdc2 kinase through wee 1 or Cdc25 [53]. The addition of inhibitor-2 (a specific protein inhibitor of PP-1, see below) to Xeno pus oocyte extracts causes premature activation of Cdc2 kinase and entry into mitosis, whereas addition of PP-1 retards mitosis [54**]. As PP-1 activity is high during interphase and reduced before mitosis (see below), these observations suggest that the PP-1-mediated dephosphorylation of unknown interphase substrates delays mitosis, perhaps while other preparations for mitosis are being completed, and that PP-1 activity must subsequently be suppressed to allow the actual initiation events to occur. .

Cell cycle-dependent changes in PP.1 localization and activity are consistent with this phosphatase having dual functions in mitotic entry and exit. The activity of PP.1 peaks during interphase, decreases near the onset of mitosis, and peaks again during mitosis when Cdc2 kinase activity is maximal [54*-l. Alterations in PP-1 localization may regulate its activity and substrate specificity, because in mammalian cells PP-1 is cytoplasmic during Gt and S phases and accumulates in the nucleus during G2 and M progression, where it is tightly associated with condensed chromosomes during mitosis [49’]. Two specific protein inhibitors of PP-1, inhibitor-l and inhibitor-2, may also modulate PP-1 activity in a cell cycle-dependent manner. The level of inhibitor-2 oscillates with the cell cycle, but peaks when PP-1 activity is required for mitotic exit [ 551. However, Cdc2 kinase may catalyze its own inactivation, because Cdc2 can phosphorylate and inactivate inhibitor2 in vitro, a reaction that activates PP-1 [56-l. Mutation of a PP-2A gene (ppu2+ ) in fission yeast results in premature mitosis [ 571, as does the inhibition of PP-2A with okadaic acid in Xenopus oocyte extracts [ 581, and PP-2A is a component of INH, which was identified as an activity that prevents activation of p34cdc2 during G2 [59*]. Thus PP-2A, as well as PP-1, appear to regulate the timing of initiation of mitosis. One way in which PP2A could effect this is by acting in a cell cycle-specific manner to maintain Thrl61 of Cdc2 in a dephosphotylated form and consequently prevent cyclin binding to Cdc2 (511. In fact, a critical concentration of cyclin is required to overcome INH activity, but okadaic acid does not enhance cyclin binding to Cdc2 [ 511, suggesting that neither PP-2A nor PP-1 regulate the interphase phosphorylation of Thrl61. A more likely but indirect mechanism by which PP-2A could prevent Cdc2 activation is by maintaining Cdc25 in a dephosphotylated form, which would block Tyrl5/‘T’hr14 dephosphotylation of CdQ. The Cdc25-specific phosphatase is sensitive to lower concentrations of okadaic acid which inhibit PP-2A [43**]. As PP-2A levels do not alter during the cell cycle [ 57,601, PP-2A activity must be tightly regulated, with a precisely timed reduction in activity occuring near the initiation of

mitosis (a decrease in the Cdc25specific phosphatase activity has been observed at this point [43**]). Although PP-2A activity is reported not to change through the cell cycle, this could reflect the use of artificial substrates in the assays [ 57,601. Tyrosine phosphorylation of PP-24 inhibits phosphatase activity [61*] and various regulatory PP-2A subunits could also modulate phosphatase activity or specificity.

Mitosis-specific

activation

of src kinase

The non-receptor tyrosine kinase ppbOc-src is a downstream target of p34cdC2 that is activated at mitosis and repressed during interphase. Phosphorylation of Tyr527 in src effects a negative regulation of kinase activity. Mitosisspecific dephosphorylation of Tyr527 is partially induced by p34cdc2-dependent phosphorylation of the src aminoterminal residues Thr34, Thr46 and Ser72 (suggesting that phosphoseryl/threonyl-src is a better substrate for a Tyr527specific phosphatase or a worse substrate for a lyr52i’specific kinase), and partially induced by a mechanism independent of p34cdc2-mediated src phosphotylation (suggesting activation of a Tyr527specific phosphatase or inhibition of a Tyr527specific kinase) [62,63*1. A receptor-like tyrosine phosphatase, PTPa, is a candidate ‘Iyr-527 phosphatase. Overexpression of PTPa in Iibroblasts induces Tyr527 dephosphotylation and persistent activation of ppGO~-~rc[64**]. However, cellular overexpression of a non-receptor tyrosine phosphatase, PTP lB, has no effect on src phosphorylation or activity [65], indicating that PTPa-mediated effects on src may be specific. PTPa could mediate the mitosis-specific, and/or the growth factor-induced, activation of src kinase. Overexpression of PTPa in fibroblasts also causes cell transformation and tumourigenesis [64**]. Thus PTPa may function as an oncogene, possibly if overexpressed as a consequence of gene translocation or amplification, or as a result of defects in the regulation of this phosphatase. Nothing is presently known about the regulation about PTPa activity.

Conclusion

Receptor-like tyrosine phosphatases (CD45, PTPa) act as upstream activators of tyrosine kinases, and a non-receptor tyrosine phosphatase (cor~screzu) acts as a downstream effector of a tyrosine kinase, indicating that such phosphatases may represent missing links in numerous transduction pathways involving tyrosine phosphosphorylation. Studies with specific inhibitors and genetic approaches are revealing unsuspected actions of the serine/threonine phosphatases in cell signalling pathways. We predict that many more instances of the critical function of both tyrosine and serine/threonine phosphatases in transduction will be elucidated, and that signalling cascades will be shown to utilize ‘cross-talk between

Protein

serine/threonine/tyrosine phosphatases and kinases as a primary mechanism of signal propagation and cell regulation.

phosphatases

in cell signalling

Pallen, Tan, Guy

sponse to light. It is aiso expressed in mushroom bodies of the central brain, where its function is unknown. 10.

STEELE F, OTOUSA JE: Rhodopsin Activation Causes Retinal Degeneration in Drosophila rdgC Mutant. Neuron 1990, 4:883-890.

11.

References

and recommended

reading

KORETWV G& PICUSJ, THOMASML, W~tss A: ‘IpsIIe Phosphatase CD45 is Essential for Coupling T-cell Antigen Receptor to the Phosphatidyl Inositol Pathway. Nature 1990, 346d6-68.

Papers of particular interest, published within the annual period of review, have been highlighted as: . of special interest .. of outstanding interest 1.

COHEN P: The Structure and Regulation of Protein Phosphatases. Annu Rev Rio&m 1989, 58:453-508.

2.

FISCHEREH, CHARUONNEAIII-1. TONKS NK: Protein Tyrosine Phosphatases: A Diverse Family of Intracellular and Transmembrane Enzymes. Science 1991, 253:401406.

3.

CAO X, i%fAHENDRAN R, GUY GR. Tm YH: Protein Phosphatase Inhibitors Induced the Sustained Transcription of the egr-1 Gene and the Hyperphosphotylation of its Gene Product. J Biol Chem 1992, 267:12991-12997.

4. ..

Gtrv GR, CAIK~‘sJ, NG SB, TAN YH: Inactivation of a Redox Sensitive Protein Phosphatase During the Early Events of TNF/IL-1 Signal Transduction. J Biol Chenz, in press. Demonstrates that TNF/IL-1 treatment of cells inhibits the activity of a .serine/threonine phosphatase. Cellular phosphatase activity towards labelled phospholHsp27 was measured by cold phosphate chases in the presence/absence of TNF/lL- I. 112r?lro assays of phosphatase activity in TNF/IL.I treated cells were also carried out. The results of both types of experiments indicate that phosphatase inhibition is an early event in TNFiIL-1 signal transduction. 5. ..

PEWINS LA, LU~~EN 1. PERKIMONN: Corkscrew Encodes a Putative Protein Tyrosine Phosphatase that Functions to Transduce the Terminal Signal from the Receptor Tyrosine Kinase Torso. Cell 1992, 4:225-236. Mutant cor%screu~emhryos have abnormally developed embryo termini. The cor&reu~gene encodes a tyrosine phosphatase with SH2 domains, and genetic analyses show that it acts in the tonso tyrosine kinase signal transduction pathway to activate downstream terminal genes. 6.

.

SHEN SH, BAV~EN L, POSNERBl, CHRETIEN I’: A Proteintyrosine Phosphatase with Sequence SimIlarIty to the SH2 Domain of the Protein-tyrosine Kinases. Nature 1991, 352:736-739.

This report discusses cloning of a novel type of tyrosine phosphatase possessing two amino-terminal SH2 domains. The SH2 domains of this phosphatase can bind to phosphotyrosine-containing proteins, including the epidemrai growth factor receptor. 7. .

PLUTLKYJ, NEELBG, ROSENBERG RD: Isolation of a Src Homology 2Containing Tyrosine Phosphatase. Proc Nat1 Acad Sci USA 1992, 89:1123-1127. Cloning of an SH2 domain-containing tyrosine phosphatase closely related to that discussed in [6*] is described. This phosphatase is highly expressed in haematopoietic and epithelial cells. 8. .

YI T. C~VEVWD JL, ISLE JN: Protein Tyrosine Phosphatase Containing SH2 Domains: Characterization, Preferential Expression in Hematopoietic Cells, and Localization to Human Chromosome 12p12-~13. Mol Cell Biol 1992, 12:836-846. This paper reports the cloning of an SH2 domain-containing tyrosine phosphatase that is closely related to the phosphatase discussed in [6*]. This phosphatase is predominantly expressed in haematopoietic cells. STEEU FR, WASHBURNT, R~ECERR, O’TOUSA JE: Drosophila retinal degeneration C (rdgC) Encodes a Novel Serine/Threonine Protein Phosphatase. Cell 1992, 69669-676. The ‘d&gene encodes a new type of setine/threonine phosphatase that may be regulated by Ca*+ binding. RdgC protein is expressed in the adult Ry visual system, where it acts in an unknown rhodopsin-initiated signaIling pathway to prevent photoreceptor degeneration in re-

KOR~KV GA, PICUSJ, SCHULTZ T, WE&~ A: Tyrosine Phosphatase CD45 is Required for T-cell Antigen Receptor and CDZ-mediated Activation of a Protein Tyrosine Kinase and Interleukin 2 Production. Pnx Nail Acud Sci USA 1991, B&2037-2041. A CWS-deficient Jurkat cell clone is used to examine signallmg defects upon activation of the TCR or stimulation of the accessory molecule CD2. Responses that are lacking in these ceUs include no increased in tracellular calcium, inositol phosphatases or tyrosine kinase acivity, and no IL-Z production, indicating that CD45 is normally required for these sigmalling events. 12.

..

13.

TRO~~RIDGEIS: ClX5.

14.

IZQUIERD~M, C~REU. DA T-cell Activation. 1992, 2:268-271.

15.

KLUSNER RD. SAMEL~~NLE: T-cell Antigen Receptor Activation Pathways: The Tyrosine ICinase Connection. Cell 1991, 64:875-878.

C&m

1991, 35:23517-23520. Trends

Cell Biol

OS’IFRGAARDHL, TROWBRIDGEIS: Negative Regulation of CD45 Protein Tyrosine Phosphatase Activity by Ionomycin in T CelIs. Science 1991. 25314231425. A Ca”+.dependent regulation of CD45 activity is suggested by the finding that treatment of murine T cells with ionomycin increases inttaceUular Ca*+ and decreases CD45 phosphatase activity. Setine phosphotyiation of CD45 also decreases, but whether this is the cause of decreased CD45 activity is unknown. 16.

..

17.

SCHRE~BERSL: Chemistry and Biology of the Immunophilins and Their Immunosuppresive Ligands. Science 1991, 25 1~283-287.

18. ..

L4u J, FARMERJD. LANE WS, FittEDMAN J, WEISSMAN I, SCHREIBERSL CaIcineurin is a Common Target of Cyclophilin CyclosporIn A and FKBP-FlC506 Complexes. Cell 1991, 66807-815. Describes key discovery of a cellular binding target for the drug immunophilin complexes CsACyP and FK506-FKBP, identification of the target as the phosphatase, cakineurin, and the finding that binding of calcineurin by these complexes inhibits Ca*+ . and caimodulin-dependent phosphatase activity. These results substantiate a ‘gain of function’ theory, whereby the drug-receptor complex modifies the Function of an intracellular target. Ltu J, ALBERTMW, WAND- TJ, Lulu\r S, ALBERGDG, BEISHAW PJ. COHENP, MACKINTOSHC, KLEE CB, XHRE~BERSk inhibition of T CeU Signaling by Immunophili~Ligand Complexes Correlates with Loss of Calcineurin Phosphatase Activity. Biochemistry 1992, 31:38&3901. The ability of the immunosuppressant Iigands CsA and FK506 and their analogues to inhibit NFAT-mediated gene transcription correlates with their ability to inhibit calcineutin phosphatase activity. The l&and-immunophilin complexes do not inhibit phosphatases-I, -2A or -2C. The i&and contains recognition elements for both the immunophilins and cakineurin. 19. ..

20.

.

9.

..

J Biol

FRUMAND4 KNEE CB, BIERERBE, BURAKOFFSJ: CaIcineurIn Phosphatase Activity in T Lymphocytes is Inhibited by PK 506 and Cyclosporin A. Proc Nat1 Acud Sci USA 1992, 89:368&3690.

Concentrations of the immunosuppmssants CsA and FK506 that inhibit IL-2 production in activated T cells also inhibit caicineutin phosphatase activity. 21.

MCKEON F: When Worlds Collide: Immunosuppressants Meet Protein Phosphatases. Cell 1991, 66:823-826.

1005

1006

Post-transcriptional 22.

SCHREIBER SL

Action 23. .

processes ImmunophiEn-sensitive Siinaling Pathways.

in Cell

WM, CORTHESY B, Bm of a T-cell Transcription

Protein binding

24.

O’KEEFE

SJ, TAh4UdA

EA

35.

..

FK-506Promoter

and CsA-sensitive by Calcineurin.

Nufure 1992, 357:692-694. Expression of the catalytic subunit of cakineutin in Jurkat cells increases the concentration of CsA and FKSO6 required to inhibit IL-2 promoter.dependent transcription. Expression of a mutant calcineunn catalytic subunit encoding a truncated phosphatase with Cal+ independent activity allows Ca2+-independent activation of the IL-2 promoter. These results indicate that calcineurln is involved in the Ca2+-dependent and immunosuppressant-sensitive activation of the IL-2 promoter.

36. .

J, KINCAID

RL,

TOCCI

Activation

of

MJ,

the

CUPSTONE NA, CRABTREE GR: ldentilication a Key Signalling Enzyme in T-lymphocyte 1992, 357:695697.

O’NEIU

27. ..

T, A~BERS MW, SCHRE~ER Sl HOHMAN RJ: Immunophilin Ligands Demonstrate Common Features of Signal Transduction Leading to Exocytosis or Transcription. Prcc Nat1 Acad Sci USA 1991, 88:62296233.

HULTSCH

CYERT

MS,

KUNI~AWA R, KAlM and CNAZ

Homologs (CNAI malian Calcineurin, Phosphatase. Prcc

D, THORNER J: Gene Products)

a Cahnodulin-regulated

Yeast

Has Mam-

of

Phosphoprotein

Nat1 Acad Sci USA 1991, 88:7376-7380. Two genes encoding related forms of cakineurin were isolated from yeast. Mutation of either gene enhances yeast sensitivity to mating pheromone-induced growth arrest, while double mutants are even more sensitive and are unable to resume growth in the presence of the pheromone. These results indicate a role of calcineurin in the recovery of yeast cells from mating response ceU cycle arrest.

28.

STEINER JP, DAU;SON TM, FOT~IHI M, Gu\rr COHEN N, SNYDER SH: High Brain Densities

philin FKBP 358:58&587. 29. .

BRA~~GAN

Colocalized

DL

with

CE, SNOW~MAN AM,

of the ImmunoNufztre 1992,

Calcineurin.

PINAULT FM: Activation

tyrosine Phosphatase Involving lipid-dependent Protein Kinases. 1991, 8866966700.

of Membrane ProteinCAMP- and Caz+/PhosphoPm

Natl

Autd

Sci

FLO~UO T, STORK PJS: G Protein Activation Hormone-stimulated Phosphatase in Human Tumor

of a Cells.

PAN MG,

Science 1992, 256:121S-1217. The hormone somatostatin stimulates tyrosine phosphatase activity in pancreatic cancer cells. Stimulation is blocked by pretreating the cells with the G-protein inhibitors pertussis toxin or non-hydrolyzable GDP, This is the first example of a G-protein-coupled phosphatase.

T, Karl Phosphorylation.

M: The Regulation Cell 1992, 70~375-387.

31.

HUNTER

32.

JACKSON SP: Regulation

Phosphotylation.

Trends

33.

MOU

T, TEBB,

.

Role

of Phosphorylation

of Transcription CeN Biol 1392,

G, SU~ANA

II,

and

ROB~T~CH

the CDC28

of

Transcription Factor

Activity

of c-Jun Activity.

kinase C activation activity of c-Jun.

NASMYM

MIUAR JBA,

Protein

Kinase

the nuclear

the dephosphotylation

Cdc25 Phosphatase.

Protein

JR, KAREN

Decreases Regulate and DNA-

M-phase Inducer: An UnCell 1992, 68:407-i10.

KUMAGA~ A, DUNPHY

WG: The Cdc25 Protein Controls Tyrosine Dephosphorylation of the Cdc2 Protein in a Cell-free System. Cell 1991, 64903914.

Demonstration that the addition of purified Cdc25 protein to Xenopus oocyte extracts or to affinity purllied pre-maturation promoting factor induces tyrosine dephosphotylation and activation of Cdc2. The Cdc25 protein is not identified as as tyrosine phosphatase, but is proposed to stimulate the activity of a CdcZ-specific tyrosine phosphatase or to inactivate an inhibitor of Cdc2 dephosphorylation.

37.

STRAUSFEU) U, L\onE JC, FESQUET D, CAVADORE JC, PICARD A, SADH~I K, RUSSEU P, DC&E M: Dephosphorylation and Activation of a p34cdcXycEn B Complex in vitro by Human

Cdc25

Protein.

Nature

1991,

351:242-245.

Demonstration that the addition of purified Cdc25 to purIIied CdcZ-cyclin B results in tyrosine and threonine/setine dephosphorylation of Cdc2 and activation of the kinase activity of Cdc2. Cdc25 is proposed to be a phosphatase specific for CdcZ-zyclin B.

38. .

DLINI’HY

Intrinsic

WG, KUMAGAJ A The Phosphatase Activity.

Cdc25

Protein

39.

GAUGER

J, SOLOMON

.

MW: Cdc25 is a Specitic Tyrosine Phosphatase Activates p34aca. Cell 1991, 67:197-211.

Contains

an

Cell 1991, 67:189196. The Cdc25 protein possesses intrinsic tyrosine phosphatase activity towards a variety of synthetic substrates, including a Cdc2 phosphopeptide. Cdc25 shares several properties with the classical tyrosine phosphatases, such as inhibition by vanadate, molybdate, zinc and alkyIatIng agents, and loss of activity upon mutation of an essential cysteine residue in the active site. MJ,

BOOHER

RN,

BAZ&N JF,

that

K~RSCHNER

Directly

Purified Cdc25 activates purified CdcZ-cyclin B and dephoshorylates TyrlS and Thrl4 of Cdc2. Sequence comparisons show that Cdc25 has regions of sequence homology with other tyrosine phosphatases. Mutation of the essential cysteine residue in the predicted active site of Cdc25 abolishes tyrosine phosphatase activity.

40.

MI~~AR JBA,

.

p8Ocdcas Mitotic Activates p34cdca 10:43014309.

MCGOWAN

CH,

Inducer Kinase

LENAERS G, JONES

R, R~I~SELL P:

is the Tyrosine Phosphatase that in Fission Yeast. EMBO J 1991,

Cdc25 induces tyrosine dephosphorylation and activation of Cdc2, and is also capable of dephosphotylating setine residues in the synthetic substrate casein. Cdc25 exhibits the characteristic responses to various inhibitors of other tyrosine phosphatases, and Cdc25 activity is abolished by mutation of its active site cysteine residue. These results indicate that Cdc25 is a tyrosine phosphatase with dual speciliciry. 41. .

LEE MS, 0% P~ICA-WORMS

that

S, Xu

H: Dephosphorylates

M, PARKER LL, D~NOGHUE

DJ, MALL!ZR JL

Cdc25 Encodes a Protein Phosphatase p34cdca. Mol Biol Cell lYY2, 3173-84.

Purified Cdc2S activates Cdc2 in Xenopus extracts and immunoprecipitates. dephosphorylates recombinant Cdc2 on tyrosine, and dephosphorylates para-nitrophenyl phosphate. Cdc25 is suggested to be the tyrosine phosphatase responsible for the activation of Cdc2 at the G2-M phase transition.

42. .

by

The first

in

S. cereofshe

at Sites That Negatively Cell 1991, 64:573584.

RUSSEU P: The

conventional

by

K: The

the

66:743758. is necessaty for

enhances

Gum K, BROYLES SS, DD;ON phatase Encoded by Vaccinia 362. demonstration

43. ..

KUMAGA~

tein

During

70:139-151.

A,

phosphatase

WG: Cycle

Regulation in Xenopus

DUNPHY

the

A Tyr/Ser Virus. Nature

JE:

of a tyrosine

2:104-108. H,

of

BOYLE WJ, %EAL T, DEFIZE LHK, ANGEL P, Wo0oGs’t-r M, HUNTER T: Activation of Protein Kinase C

Phosphorylation its DNA-binding

USA

Treatment of cells with activators of serine/threonine protein kinase A and C or with inhibitors of setine/threonine phosphatases results in the activation of a membrane tyrosine phosphatase that is part of a high molecular weight protein complex. A model is proposed where serine/threonIne phosphotylation of a regulatory component in the complex modulates tyrosine phosphatase activity.

30. .

.

.

Overexpression of the calcineunn catalytic subunit in Jurkat cells makes these cells more resistant to the immunosuppressants CsA and FK506 and enhances NFAT- and NFILZA-dependent transcription. The authors propose that calckteurin regulates the nuclear translocation of NFAT.

26.

34.

Interleukin-2

of Calcineurin as Activation. Nature

Nuclear Import SwrS. Cell 1991,

Cell cycle-dependent dephosphotylation translocation of SWIS.

RJ, CRABTREE GR Nuclear Factor Blocked by FK506 and Cyclosporin A. Nufure 1991, 352:803807. Activation of NFAT requires the Ca a+ -dependent translocation of a constitutive cytoplasmic subunit to the nucleus, where it combines with a PMA-induciblenuclear subunit to form an active transcription factor. The cytoplasmic component of NFAT appears to be T cell specific. The Immunosuppressants CsA and FKSO6 block nuclear translocation.

25. ..

FUN&AN Association

Cell Cycle-regulated Transcription Factor

Protein* Phosphatase Cell 1992, 70:365-368.

Cell

with

of

Protein Phos1991, 350:359dual specificity.

the Cdc25 ProExtracts. Cell lYY2,

Protein The Cdc25 protein undergoes cell cycle-dependent phosphorylation and dephosphotylation. which regulate its tyrosine phosphatase actlvity. Near the onset of mitosis, the activity of a Cdc25-specilic kinase increased and that of a Cdc25-specific phosphatase decreased, resulting in extensive phosphotylation and activation of Cdc25. The activity of the Cdc25-specific phosphatase is higher during interphase, resulting in dephosphotylation and reduced activity of Cdc25. This work is a good demonstration of how the concerted regulation of a kinase phosphatase system can modulate the phosphotylation state and activity of a target substrate. GA~AK~ONOVK, BEACHD: Specific Activation of Cdc25 Tyrosine Phosphatases by B-type Cyclins: Evidence for Multiple Roles of Mitotic Cyclins. Cell 1991, 67:1181-1194. The in r&o phosphatase activity of Cdc25 is stimulated about live.fold in the presence of B-type cyclins, but not cyclins A or D. Maximum actlmtion of Cdc25 requires a stoichiometric amount of cyclin B. This suggests Cdc25 may interact with the CdcZ-cyclin B complex during catalysis.

phosphatases

in cell signalling

Pallen, Tan, Guy

55.

BRAUT&AN DI, SU~‘WOOJ, IAFjBfi J-C, FERNANDU.4 m NJC: Cell Cycle Oscillation of Phosphatase Inhibitor-2 in Rat Fibroblasts Coincident with p3@C2 Restriction. Nature 1990, 33474-78.

56.

Activation of Type-l Protein Phosphatax FEBS Len 1992, 304:211-215. with inhibitor-2 can be activated by the in inhibitor-2 by Cdc2 kinase.

VU-MORUZZI E: by Cdc2 Kinase. Inactive PP.1 complexed h-o phosphorylation of .

57.

44. ..

K~NOSHITAN, OHKURA H, YANAGIDA M: Distinct, Essential Roles

of

Type

1 and

2A

Protein

Phosphatases

in

the

Control of the Fission Yeast Cell Division Cycle. Cell 1990, 63:405-415. 58.

Flux M-A, COHEN, P, ~ENTI E: Cdc2 Hl Kinase is Negatively Regulated by a Type 2A Phosphatase in the Xenopus Early Embryonic Cell Cycle: Evidence from the Effects of Okadaic Acid. EMBO J 1990, 9675-683.

45. ..

JEWS

C, BEACH D: Oscillation of MPF is Accompanied by Periodic Association Between Cdc25 and CdcZ-Cyclin B. Cell 1992, 68~323-332. CdcZcyclin B associateswith Cdc25 in a cell cycle-dependent manner, with maximal association during M phase at the time of peak Cdc2 kinase activity. Association is regulated and may be required for the dephosphotylation of Cdc2.

LEETH, SOLOMONMJ, MUMBYMC, KIRSCHNER MW: INIi, a Negative Regulator of MPF, is a Form of Protein Phosphatase 2A Cell 1991, 6441-23. The demonstration that PP-2A is a component of INH, an activity ln Xenopw oocytes that inhibits activation of the p34C*c+yclin complex. The in vitro inactivation of p3&*c2 kinase correlates with its dephosphotylation by INH.

46.

OHKURA H, WNOSHITAN, MNATANI S. TODA T, YANAGIDAM: The Fission Yeast dis2+ Gene Required for Chromosome Disjoining Encodes One of Two Putative Type 1 Protein Phosphatases. Cell 1989, 57:w7-1007.

60.

47.

DOONAN JH, MORRISNR: The bimG Gene of Aspergillus niduluns, Required for Completion of Anaphase, Encodes a Homolog of Mammalian Phosphoprotein Phosphatase 1. Cell 1989, 57: 987-996.

48.

AXTON

JM, D~MBRADI V. COHEN PTW, GLOVERDM: One of the Protein Phosphatase 1 &enzymes in Drosophila Is Essential for Mitosis. Cell 1990, 63~33-46.

49. .

FERNANDEZ A, BRAUTICANDL, LAMB NJC: Protein Phosphatase Type 1 in Mammalian Cell Mitosis: Chromosomal Localization and Involvement in Mitotic Exit. / Ceil Biol 1992, 116:1421-1430. lmmunofluorescence studies of PP.1 localization during the cell cycle combined with micro-injection of PP.1 or neutralizing anti-PP.1 antibodies demonstrate a nuclear role for PP.1 in late mitosis. CIRCA T, IABB~ J-C, DEVAULT A, FESQUET D, CAPOM’ J-P, 50. . CAVAWRE J-C, LE BOUFFANT F, Do& M: Dephosphorylation of Cdc2 on Threonine 161 is Required for Cdc2 Kinase Inactivation and Normal Anaphase. EMBO J 1992, 11:2381-2390. An investigation of the steps in Cdc2 inactivation. Cyclin degradation and dephospholylation of Thrl61 in Cdc2 are required for complete inactivation and mitotic exit. Demonstrates the essential role of a protein phosphatase. perhaps PP-1, in mitotic exit. 51.

DUCO~IMUNB, BR~MBILIAP, Flux M-A, FIUNZABR JR, KARSENTI E, DRAETTAG: Cdc2 Phosphorylation is Required for its Interaction with Cyclin. EMBO / 1991. 10:3311-3319.

52.

Gouu, KL, MORENOS, OWN DJ, SAZERS, NURSEP: Phosphorylation at Thr167 is Required for Schizosuccbaromyces pombe p34C*c2 Function. EMBO J 1991, 10:3297-3309.

53.

BOTHER R, BEACHD: Involvement of a Type 1 Protein Phosphatase Encoded by bwsl+ in Fission Yeast Mitotic Control. Cell 1989, 57:1009-1016. WAU(ER DH, DEPAOU-ROACHAA, MUER JL: Multiple Roles 54. .. for Protein Phosphatase 1 in Regulating the Xenopus Early Embryonic Cell Cycle. I~IoI Biol Cell 1992, 3687-698. The first demonstration that PP.1 activity fluctuates with the cell cycle, probably due to the use of an appropriate cell cycle substrdte, CdcZ-phosphorylated histone Hl. The authors also provide biochemical evidence that PP.1 is involved in regulating the timing of initiation of mitosis by manipulating the level of PP.1 activity in oocyte extracts.

59. .

RUEDIGERR, HOOD JEVW, MUMBY M, WALTERG: Constant Expression and Activity of Protein Phosphatase 2A in Synchronized Cells. Mol Cell Biol 1991, 11:4282-4285.

61. .

CHEN J, MARTIN BL, BRAUT~GANDL: Regulation of Protein Serine-threonine Phosphatase Type-u\ by ‘Qrosine Phosphorylation. Science 1992, 257~1261-1264. The p6Ov.s~, p56lck, epidermal growth factor receptor and insulin receptor kinases can caee tyrosine phosphotylation of PP-2A with a consequent inhibition of PP-2A activity. Phosphorylation occurs on a carboxyl-terminal tyrosine residue within a sequence not shared by PP.1. Auto-dephosphorylation of PP-2A may ensure that inhibition of phosphatase activity is transient. 62.

SHENOYS, CHOI J-K, BAGRODLAS, COPEL~NDTD, MUER JI, SHAU.OWAYD: Purified Maturation Promotion Factor Phosphorylates pp6OC-Smat the Sites Phosphorylated During Fibroblast Mitosis. Cell 1989, 57763774.

SHENOY S, CHACUARAMPIL 1, BAGRODLA S, m P-H, SHAU~WAY D: Role of p34cdc2-mediated Phosphorylations in Two-step Activation of pp6OC-SrCDuring Mitosis. Proc Nut1 Acud Sci USA 1992, 89:7237-7241. At mitosis, the CdcZ-catalped serine/threonine phosphotylation of pp~c-src enhances the subsequent dephosphorylation of Tyr527 in pp6OC-Srcand activation of the src kinase. 63. .

ZHENGXM, WANG Y, PAL~ENCJ: Cell Transformation and Activation of pp6OC-SrCby Overexpression of a Protein Tyrosine Phosphatase. Nulure 1992, 359336339. The receptor-like tyrosine phosphatase, PTPa, can function as an up stream actimtor of the tyrosine kinase p*SK by mediating dephosphorylatlon of Ty1-527in pp6OC~s~.Overexpression of PTPa results in cell transformation and tumourigenesis, providing the first evidence that a tyrosine phosphatase has an oncogenic capability.

64. ..

65.

WOODFORD-THOR TA, RHODES JD, DEVONJE: Expression of a Protein Tyrosine Phosphatase in Normal and US~XGWSformed Mouse 3T3 Fibroblasts. J Cell Bioll992, 117:401-%14.

CJ Pallen, Cell Regulation laboratory, Institute of Molecular and Cell Biology, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511, Republic of Singapore.

YH Tan and GR Guy, Signal Transduction laboratory, Institute of Molecular and Cell Biology, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511, Republic of Singapore.

1007