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Biochemical Society Transactions ~ ~~ ~~~ Important roles for novel protein phosphatases dephosphorylating serine and threonine residues 884 Patricia T. W. Cohen Medical Research Council Protein Phosphorylation Unit, Department of Biochemistry, The University of Dundee, Dundee DDI 4HN, U.K. Introduction Phosphorylation of proteins on serine and threonine residues is a key mechanism in the regulation of a wide variety of cellular functions, including cell division, hormonal action and signalling from the plasma membrane to the nucleus [ 1-01. The revsibility of protein phosphorylation requires that dephosphorylation as well as phosphorylation processes must be elucidated to understand the regulation of cellular functions by this mechanism fully. Four major protein phosphatase (PP) catalytic subunits were identified originally by enzymic criteria in mammalian cells, and termed PP1, PP2A, PP2H and PP2C [4] and isoforms of each of these phosphatases were identified subsequently by molecular cloning. These studies also showed that PP1, PP2A and PP2H are related in structure, while PP2C does not contain the regions predicted to be essential for the catalytic activity of the PPl/PP2A/PP2H family [ S , 61. Size of the PP I /PPZA/PPZB family Over the last few years, several novel protein phosphatases belonging to the PP 1/PP2A/PP2H family have been identified from their cDNAs. These include PPX [7] in mammals, PPV [6] and PPY [S] in Drosophila melanogaster, and PPZl [6, 9, 101 and PPZ2 191 in Saccharomyces cerevisiae. Others have been identified by the analysis of a mutant of the protein SIT4 in S. cerevkiae [ 111 and a mutant of the protein rdgC in DrosophiZa [ 121. PCR studies were initiated [ 131 to estimate the number of different catalytic subunits in this family of protein phosphatases. Oligonucleotides constructed to sequences conserved between mammalian and bacteriophage I protein phosphatases [ S ] were used with genomic DNA from S. cerevisiae, D. melanogaster and Homo sapiens. Sequence determination of the PCR fragments identified both known and novel protein phosphatases in S cerevisiae and Drosophila and novel protein phosphatase genes (or pseudogenes) in man. In Drosophila, seven novel and two protein phosphatase genes that were already known were identified by this procedure. The results suggest Abbreviation used: I T , protein phosphatase; MAP kinase, mitogen-activated protein kinase. Volume 21 that, if we detected all eight protein phosphatase genes that were known in Drosophila at that time, then we should have identified 28 novel protein phosphatase genes, bringing the total number of protein phosphatase genes in this species to 36. Since the human genome may encode tenfold the number of proteins encoded by the Drosophila genome and the complexity of protein phosphorylation undoubtedly increases in higher eukaryotes, there may be more than 300 genes for the catalytic subunits of this family of protein phosphatases in mammals. PP1, PP2A and PP2H catalytic subunits all bind at least two different regulatory subunits, indicating that >900 genes or approx. 1% of human genes may encode this family of protein phosphatases. Protein phosphatases in the P P l and PP2A subfamilies identified from full-length cI )NAs or genes are shown in Table 1 for the three species: man, Drosophila and S. cerevisiae. T o determine whether the novel protein phosphatases play distinct roles from those of PP1 and PP2A, we have used several approaches to investigate their functions. The results point to specific and key roles for mammalian PP4 (PPX), Drosophila PPV and S cerevisiae PPZ 1 and PPZ2. PP4 (initially termed PPX) [ 141 Rabbit PP4 was expressed from its cDNA in the baculovirus/insect cell system. Although mostly insoluble, the 10% that was soluble and active was purified partially to remove contaminating insect cell protein phosphatases. Examination of its substrate specificity showed that PP4 dephosphorylates serine and threonine residues. As with PP2A, PP4 preferentially dephosphorylates the a subunit rather than the p subunit of phosphorylase kinase, and is unaffected by inhibitor-1 and by inhibitor-2. PP4 and PP2A were also inhibited similarly by the tumour promoter okadaic acid and the liver toxin microcystin (the ICirrvalues for okadaic acid were 0.07 nM for PP2A and 0.2 nM for PP4, while for microcystin they were 0.2 nM and 0.8 nM respectively). However, when PP4 and PP2A catalytic subunits were matched for phosphorylase phosphatase activity, PP4 had a similar activity towards the synthetic peptide RRATj’P-VA, but was less active Signalling from the Plasma M e m b r a n e to the Nucleus Table I Protein phosphatases in the subfamilies PPI and PPZA The novel enzymes in each family are indicated. References for the sequences of PPI and PPZA isoforms are given in [ 131. References for novel phosphatase sequences are PPY [8]. PPZ I [6, 10, 241 PPZ2 [9. 291, PPQ 1291, mammalian PP4 [ 141, Drosophria PP4 [30],PPV [ 181, SIT4 [ I I ] and PPG [3 I ] Mammals Drosophilo S PP I a PPlg PPI y PPI 878 PPI 96A PPI 13C PPI 9c PPY PP I (DIS2) cerevisioe PP I subfamily Established Novel PPZ I PPZ2 PPQ PPZA subfamily Established PP2Aa PPZAB PPZA Novel PP4 (PPX) PP4 (PPX) PPV PPH2 I PPH22 PPH3 SIT4 PPG than ”2.4 towards all other substrates tested (casein, histone EI 1, caldesmon. €IMG-I). The I’P2A:PP4 activity ratio varied from 1 : l to 13:l with different substrates, indicating that the specificities of the two enzymes are distinct. Furthermore, despite 65% amino acid sequence identity to PP2A, I’P4 did not bind the 65 kDa regulatory subunit of I’P2A. Evidence pointing towards a specific function for PP4 came from immunofluorescent localization studies using biotinylated, affinity-purified antibodies against the PP4 protein, coupled to an avidin-fluorescein stain. These experiments demonstrated that although PP4 was present in the cytoplasm and more strongly in the nucleus of human cells, it localized intensely to the centrosomes. The localization was judged to be specific since it could be blocked by pre-incubation of the antibody with excess PP4. An identical localization pattern was obtained using an antibody raised against a synthetic peptide corresponding to amino acids 287-305 of PP4. The centrosome is one of the microtubuleorganizing centres of the interphase cell, and at mitosis it duplicates to form the spindle-pole bodies. Localization of PP4 was observed at the centrosomes of interphase cells and at all stages of mitosis except telophase. Therefore, at telophase, either the PP4 epitope must have an altered conformation or PP4 must be released from the centrosomes. Since telophase is the point at which the mitotic spindle disappears and the interphase network of microtubules reforms, PP4 may be involved in this process. Examination of the localization of PP4 at higher magnification revealed that it was absent from the middle of the centrosome, but it co-localized with antibodies that are known to detect the pericentriolar material, the region thought to be responsible for initiating the growth of microtubules. Several studies have shown that a protein phosphatase sensitive to nanomolar concentrations of okadaic acid is required for microtubule nucleation, and it has been assumed to be PP2A [ 15, 161. However, affinity-purified antibodies to PPZA localized predominantly to the cytoplasm, and were not evident at the centrosomes in interphase or in dividing cells [ 141. It can be inferred therefore that PP4, rather than PP2A, may regulate microtubule nucleation. The signal(s) that regulate PP4 activity is not known, but one possibility is the cdc2-cyclin A complex, which has recently been shown to initiate microtubule growth in vitro [ 171. Drosophilu PPV PPV [18] was identified from a cDNA in a Drosophila head library, but subsequent studies showed that PPV mRNA was only expressed at very low levels in adult tissues, while it was abundantly expressed in the early embryo. In sztu hybridization studies on Drosophih embryos around the time of cell formation (nuclear-division cycle 14) showed that PPV mRNA was localized predominantly at the periphery of the embryo, in the region undergoing cellularization. The PPV protein, detected with affinity-purified antibodies against a PPV-specific peptide also localized to the cytoplasm of cells at the cortex. No PPV could be detected in the nucleus. The substantial rise in PPV occurring transiently over the cellularization period is the time at which zygotic transcription rises and the nuclear-division cycle changes from one in which mitosis rapidly follows DNA synthesis, to the more characteristic cell cycle of eukaryotes that includes G1 and G2 phases, suggesting that PPV may be involved in one or more of these processes. I993 Biochemical Society Transactions Since the amino acid sequence of PPV is most similar to that of SIT4 in S. cermzi-iae (65% identity), it was pertinent to test whether SIT4 and PPV might be homologues. SIT4 regulates the transcription of a number of genes, including G1 cyclins [ 191, and, in certain genetic backgrounds, an allele of SIT$ sit4-1 02, causes a temperature-sensitive cell-cycle arrest in late G I [20]. Transformation of this mutant with PPV cDNA, placed under the control of the alcohol dehydrogenase promoter, allowed growth at the restrictive temperature. In contrast, cDNA for other protein phosphatases under the same promoter did not rescue sit4-102. Proof that PPV could perform all the in vivo functions of SIT4 came from the demonstration that PPV cDNA, when under the SIT4 promoter, could completely replace the S I T 4 gene in S. cerevisiae. The large increase in PPV occurring at the time when zygotic transcription increases, and the demonstration that PPV is the functional homologue of SIT4, indicate a role for PPV in the regulation of transcription. However, there is, as yet, no evidence for a rise in the transcription of any G1 cyclin in the early Drosophila embryo. SIT4 has also been shown to be essential for bud emergence in S. cerevisiae, and a parallel role for PPV in Drosophila could be in the formation of cells in the DrosophiZu embryo at nuclear-division cycle 14. The mRNA for the genes serendipi& and nullo, which are required for cellularization, rise transiently [2 11, and it may be that their transcription is under the control of PPV. Recently, ppe 1, the probable Schkosuccharomyces pombe homologue of PPV and SIT4, has been identified [22]. Deletion of the ppel gene causes arrest of cells at G2, rather than G1 as seen in S. cerevkzize. Other workers identified ppel as a suppressor of the mutant piml, in which mitosis is uncoupled from DNA synthesis [23]. These analyses implicate ppel in a process in the G2-M transition that may couple DNA synthesis and mitosis in S. pombe. Therefore, it may be that the PPV/SIT4/ppe 1 phosphatase is involved both in the G1-S and G2-M check-points. A role in the cell cycle suggests that the activity of PPV may be tightly regulated. Comparison of the PPV and SIT4 sequences revealed amino acids that were identical in these two protein phosphatases, but were different from those in other known protein phosphatases. The only section where these were more than just isolated amino acids was in the N-terminal region. Therefore, a chimeric construct (designated pADH V: 13V) was made, in which the N-terminal 5 5 amino acids of Volume 21 PPV were attached to the catalytic domain of a Drosophila isoform of PP1 (PP1-13C) and placed under the control of the alcohol dehydrogenase promoter. Rescue of sit4-102 was achieved by transformation with pADH V:13C but not by pADH 13C:V in which the N-terminus of PP1 was attached to the catalytic domain of PPV [8]. It is remarkable that the N-terminal region of PPV (a PP2A-like phosphatase) could effect the rescue of the SIT4 mutant when attached to a PP1 catalytic region. These results identify the N-terminal 5 5 amino acids of PPV as being essential for PPV function. This domain is unlikely to target PPV to a particular location within the cell, since the immunofluorescent localization studies described above showed a diffuse cytoplasmic staining for PPV. A more plausible explanation is that the N-terminal domain is involved in binding to regulatory subunits, which may influence the specificity of the catalytic domain and/or control its activity in response to incoming signals. S. cerevisiae P P Z l and PPZ2 [24] Although PPZl and PPZ2 were originally identified in a commercial rabbit brain cDNA library (Clontech), subsequent analyses demonstrated that they did not encode brain phosphatases but novel S. cereviszize enzymes [9]! Both phosphatases contain a catalytic domain that is preceded by a long Nterminal domain, rich in serine and asparagine in PPZ1, and serine and arginine in PPZ2. Although the N-terminal domains are only 43% identical to each other, the catalytic domains are 93% identical, indicating that these two phosphatases are likely to have similar or overlapping functions. To determine their cellular roles, deletions were made in the PPZl and PPZ2 genes in regions encoding the conserved motifs predicted to be essential for catalytic activity. Mutant strains disrupted in either or in both genes, showed an enlarged cell size when examined under the microscope in stationary phase. Analysis by light scatter measurements in a fluorescence-activated cell sorter confirmed this size difference and showed that the mutant ppzlppz2 cells could be restored to normal or near normal size by the inclusion of 1 M sorbitol in the growth medium. The integrity of the cell wall and plasma membrane were assessed by measuring the release of RNA from cells labelled with ['Hluridine. Although little difference between wild-type and mutant cells was observed at 28"C, cell lysis was increased in ppzl and ppzlppz2 cells at 37°C compared with wildtype cells. In addition, lysis was slightly increased in ppz2 cells and markedly elevated in ppzl and Signalling from the Plasma Membrane t o the Nucleus ppzlppz2 strains in the presence of caffeine at 28°C. 1 M sorbital partially suppressed the lytic phenotY Pe. Several protein kinase mutants give rise to a lytic phenotype. Conditional mutants of the protein PKCl (protein kinase C1) display a cell-cyclespecific lysis defect [25,261 that can be suppressed by four different protein kinases. Genetic analyses demonstrated their order of action to be PKCl +BCK1 -MKKl/MKKZ-MPKl where MKKl and MKK2 are related to mammalian mitogen-activated protein (MAP) kinase kinases and MPKl to mammalian MAP kinase. Strains carrying a deletion of RCK1, a double deletion of MKKl and MKK2 or a deletion of MPKl all show a temperature-sensitive cell lysis defect that can be suppressed by osmotic stabilizers [27]. Mutants of two other protein kinases, PRS2 and HOG1, which are involved in regulating the accumulation of intracellular glycerol in response to extracellular osmolarity changes, also show cell lysis defects, but, in contrast to PPZ disruptants, their lytic phenotypes are not suppressed by sorbitol [28].PPZl and PPZ2 are therefore more likely to function in the PKCl pathway than in the PHS2 pathway. Since the phenotype of increased susceptibility to cell lysis of the PPZ mutants is similar to that of BCKl (bypass of protein kinase C), MKKlIMKK2 and MPKl deletion mutants, it appears more likely that PPZ might act in concert with this kinase cascade rather than reverse it. Hy analogy to the regulation of glycogen metabolism by insulin in mammalian skeletal muscle, where PP1 is activated by a protein kinase that is linked to the MAP kinase cascade, PPZl/PPZ2 might be phosphorylated and activated by a component of the yeast PKCl /MAP kinase cascade. T h e serine rich N-terminal domains of PPZl and PPZ2 contain many phosphorylation sites for MAP kinase, protein kinase C and other protein kinases [24]. The exact mechanism by which PPZl and PPZ2 maintain cell integrity is not known, but it could involve cell wall construction, organization of the cytoskeleton and/or osmosensing. Several lines of evidence suggest that PPZl and PPZ2 may be membrane-bound. Both N-termini possess good consensus sequences for myristoylation, which would favour a membrane location. Two short sequences present in the N-terminal domains of both PPZl and PPZ2 are similar to short sections of a yeast osmotic growth protein and to desmoplakin respectively, both of which are located at or near the cell membrane. It is therefore plausible that PPZl and PPZ2 may dephosphory- late proteins at or near the surface of the cell membrane [24]. However, disruption of PPZl and PPZ2 in a certain genetic background can affect cell growth (M. X. Chen and P. T. W. Cohen, unpublished work) indicating that PPZl and PPZ2 could carry out other functions, and perhaps also prevent cell lysis by regulating the transcription of the enzymes involved in maintenance of cell integrity. In conclusion, PCR studies indicate that the PPl/PPZA/PPZB family is much larger than previously supposed, and analysis of phosphatases related to PP1 and PPZA have revealed that four of these novel enzymes PP4, PPV, PPZl and PPZ2 play key roles in cellular regulation that are distinct from those of PP1 and PP2A. This work was supported by the Medical Research Council, London and by the Cancer Research Campaign. 1. Cohen, P. (1992) Trends Biochem. Sci. 17,408-413 2. Meek, D. W. and Street, A. J. (1992) Biochem. J. 287, 1-15 3. Yanagida, M., Kinoshita, N., Stone, E. M. and Yamano, H. (1992) in Regulation of the Eukaryotic Cell Cycle (J. Marsh, ed.), pp. 130- 146 John Wiley & Sons, Chichester 4. Cohen, P. (1989) Annu. Rev. Bichem. 58,453-508 5. Cohen, P. T. W. and Cohen, P. (1989) Riochem. J. 260.93 1-934 6. Cohen, P. T. W., Hrewis, N. D., Hughes, V. and Mann, D. J. (1990) FEBS Lett. 268, 355-359 7. da Cruz e Silva, 0. H., da Cruz e Silva, E. F. and Cohen, P. T. W. ( 1 988) FEBS Lett. 242, 106- 110 8. Dombradi, V., Axton, J. M., Glover, Ll. M. and Cohen, P. T. W. (1989) FEBS Lett. 247, 391-395 9. da Cruz e Silva, E. F., Hughes, V., McDonald, P., Stark, M. J. and Cohen, P. T. W. (1991) Hiochim. Biophys. Acta 1089,269-272 10. Posas, F., Casamayor, A., Morral, N. and Ariiio, J. (1992) J. Biol. Chem. 267, 1 1734- 11740 11. Arndt, K. T., Styles, C. A. and Fink, G. R. (1989) Cell 56,527-537 12. Steele, F. R., Washburn, T., Kieger, R. and OTousa, J. E. (1992) Cell 69,669-676 13. Chen, M. X., Chen, Y. H. and Cohen, P. T. W. (1992) FEBS Lett. 306, 54-58 14. Rrewis, N. D., Street, A. J., Prescott, A. R. and Cohen, P. T. W. (1993) EMBO J. 12,987-996 5. I’icard, A,, Capony, J. P., Brautigan, D. I,. and Dorke, M. (1989)J. Cell Biol. 109, 3347-3354 6. Rime, H., Huchon, D.. Jessus, C., Goris, J., Merlevede, W. and Ozon, K. (1990) Cell Differ. Dev. 29, 47-58 7. Buendia, B., Draetta, G. and Karsenti, E. (1 992) J. Cell Riol. 116, 1431-1442 8. Mann, D. Dombradi, V. and Cohen. P. T. W. (1993) EMBO J., in the press J.? I993 887 Biochemical Society Transactions 888 19. Fernandez-Sarahia, M. J., Sutton, A., Zhong, T. and Arndt, K. T. ( 1992) Genes Dev. 6 , 2 4 17-2428 20. Sutton, A., Immanuel, I>. and Arndt. K. T. (1991) Mol. Cell. Hiol. 11,2133-2148 21. Kose, I,. S. and Wiesehaus. E. (1992) Genes Dev. 6, 1255-1268 22. Shimanuki, M., Kinshita, N., Ohkura, H., Yoshida, T., Toda, T. and Yanagida, M. (1993) Mol. Biol. Cell 4, 303-3 13 23. Matsumoto, T. and Beach, I). (1993) Mol. Cell. Hiol. 4,337-345 24. Hughes, V., Muller, A,, Stark, M. J. R. and Cohen. 1'. T. W. (1993) Eur. J. Hiochem. 216,2h0-270 25. Levin, 1). E. and Hartlett-Heubusch, E. (1992) J. Cell Hiol. 1 16, 122 1- 1229 20. I'aravicini, G., Cooper, M.. Friedeli, I+ Smith, 1). J., Carpenter, J.-I,., Klig, 1,. S.and I'ayton, M. A. (1002) Mol. Cell Hiol. 12, 4896-4005 27. Errede, H. and Levin, L). E. (1 993) Curr. Opin. Cell Hiol. 5, 254-260 28. Hrewster, J. I,., de Valoir, T., Dwyer, N. l).,Winter, E. and Gustin, M. C. ( I 993) Science 259, 1760- 1762 29. Chen, M. X., Chen, Y. H. and Cohen, 1'. T. W . (1003) Eur. J. Hiochem., in the press 30. Hrewis, N. 1). (1992) 1'h.D. thesis, llundee llniversity 31. I'osas, F.?Clotet, J., Muns, M. T., Corominas, J. A., Casamayor, A. and Ariiio, J. (1003) J. Hiol. Cheni. 268, 1349-1 354 Received 4 August 1993 Role of p2 I ras in growth factor signal transduction Boudewijn M.Th. Burgering, Gijsbertus J. Pronk, Jan Paul Mederna, Loesje van der Voorn. Alida M. M. de Vries Smits, Pascale C. van Weeren and Johannes L. Bos Laboratory of Physiological Chemistry, Utrecht University, 352 I GG Utrecht, The Netherlands Introduction r . I he family of human rus proto-oncogenes consists of three closely related genes: 11-rus, K-rus and N-rus [ 1 I. They encode homologous proteins of molecular mass 21 kI)a (p21r"')9located at the inner side of the plasma membrane. The rus proto-oncogenes acquire their oncogenic potential when point mutations occur at amino acid positions 12, 13 or 61, resulting in a single amino acid substitution in the protein [ 21. T o understand the consequences of these oncogenic mutations, it is crucial to understand the role that normal ~ 2 1 " ' fulfils in cell growth and differentiation. Regulation of p2 I ras activity The notion that p21"' can bind the guanine nucleotides G T P and CLIP is central to our understanding of the function of p2lras in growth factor-induced signal transduction. Through sequential binding of (;I>P and GTP, p21"' can function as a molecular switch in signal transduction [ 3, 41. Microinjection of p21"' proteins bound to CLIP or G T P has demonstrated that p2 1'"'-GI>P is biologically inactive. and hence represents the 'off state, Abbreviations used: EGF. epidermal growth factor; EKK2, extracellular-signal-regulated kinase 2; GAP, WI'ase-activating protein, GKF, guanine-nucleotide releasing factor; MAP kinase. mitogen-activated protein kinase; I'DGF, platelet-derived growth factor; I'KC, protein kinase C; I'MA, phorbol 12-myristate 13-acetate. Volume 2 I whereas p2 1 "'--GTP is biologically active, and thus represents the 'on' state of this switch. Two classes of accessory proteins are involved in the regulation of GDP/GTP binding to p21"". Firstly, the dissociation of GDP from p21"' occurs very slowly, and is catalysed by the interaction of p21"' with guanine-nucleotide releasing factors ((;KFs). Some of these proteins showing specificity towards p2 1 have been identified. Of these, p 140';"'" appears t o be exclusively expressed in brain tissue [ 51, whereas two others (mSos1 and mSos2) appear t o be ubiquitously expressed [6]. Once GTP is bound to p21"', a transient interaction is thought to occur between p2 1 ""-GTP and its effector molecule. The effector-binding region of p2 1"" has been determined by genetic methods and spans roughly the region amino acid 30-40 [7]. Although the identity of the p2 1"I' effector molecule is still elusive, recent results indicate that this may be the raf-1 kinase. In vitro association of raf-1 kinase with p21""GTP has been demonstrated [XI, and by using the yeast two-hybrid system it has been shown that this interaction may also occur in azvo 101. Furthermore, genetic evidence suggests that raf- 1 kinase acts downstream of p21"" [lo]. Eventually, after activation of downstream signalling, GTP on p2 1rr'' will be hydrolysed. The protein p2 1'",' itself displays a low GTPase activity, which is enhanced upon interaction with GI'Paseactivating proteins (GAPS), such as p120';'"' and neurofibromin. It is still not clear whether GtZI's, as "I'