Stathmin: a relay phosphoprotein for multiple signal transduction?

TIBS 16 - AUGUST1991

EXTRACELLULAR REGULATORY SIGNALS A

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Stathmin and the regulation of cell proliferation, differentiation and function.

correlated to the physiological, multihormonal regulation of the cell's differentiated function. In cultured embryonic neurons, phosphorylation of stathmin is enhanced by the tumor promotor TPA and by the cAMP-stimulating agent forskolin, as well as by neurotransmit-

ters like VIP and dopamine, presumably in correlation with the regulation of neuronal activity~2 and possibly also neuronal development or maturation ~3. In adult mouse brain, where neurons are mature and normally active, stathmin is proportionally much more phosphorylated than in embryonic brain~2.%

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Rgme 2 A composite representation of the two-dimensional pattern of stathmin forms. The various unphosphorylated (c¢0, [50) and phosphorylated (*) forms of stathmin are represented as they migrate on two-dimensional electrophoresis gels. Numbers indicate the increasingly phosphorylated states of the c¢ and [3 isoforms (o¢1--¢x3,1~1-~3) and of the two sets '16' and '17'. N1-N2 and P1-P3 designate the unphosphorylated and phosphorylated 19 kDa spots, respectively. 302

indicating again that the developmental state of the cell, as well as its physiological environment, may determine the phosphorylation of stathmin. Phosphorylation of stathmin is also related to the regulation of cell proliferation and differentiation. For example, in muscle cells, agents like TPA and the growth factors EGF and FGF that promote the proliferation of myoblasts, also stimulate the phosphorylation of stathmin ~4,~5.Similarly, phosphorylation of stathmin isoforms is associated with the stimulation of cell proliferation during activation of T lymphocytes through the CD3 and CD2 mltigens7'16. On the other hand, phosphorylation of stathmin has been related to the inhibition of cell proliferation induced by TPA in peripheral blood lymphocytes in their rapidly proliferating phase ~7. A deficient phosphorylation of stathmin even seems to be associated with the impaired down-regulation of DNA synthesis in T cell leukemic lymphoblasts ~8 and with the increased expression of N-myc in neuroblastoma tumors s. Stathmin phosphorylation also appears to be related to the regulation of cell differentiation, for example the NGFinduced neuron-like differentiation of PC12 pheochromocytoma cells% or the TPA-induced differentiation of human promyelocytic HL-60cells4. Thus, phosphorylation of stathmin has been shown to occur, even within a given cell type, in response to a variety of signals that activate distinct second messenger pathways and regulate cell proliferation, differentiation or cellspecific functions. Stathmin migrates as several unphosphorylated and phosphorylated spots 2,4,2°,u on two-dimensional polyacrylamide gels (Fig. 2). At least two basic 19 kDa isoforms, a and [3, have clearly been identified 2°. Their unphosphorylated forms, a0 and [~0,each yield at least three more acidic, increasingly phosphorylated forms, al-u3 and [31-[33, which also migrate as several complex spots (PI-P3) with slightly increasing apparent Mr (19-20 000). The two isoforms are both encoded by a single cloned cDNA22 and thus differ only by co- or post-translational modifications, the nature of which is still unknown. Two additional sets of two-dimensional PAGE phosphorylated spots, designated respectively '16' and '17' (Refs 9,10,12,19) [or pp21 and pp23 (Refs

TIBS 16 - AUGUST 1C91

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Rgure 3 Comparison of the amino acid sequencesof stathmin (ST) and SCGIO(SCG).Numberedarrowheadspoint to the predictedphosphorylation sites for the cAMP-dependent(16,46,63) and prolinedependent(25,38) protein kinases. The shaded area shows the conservationof the a-helical domain, containingthe heptadrepeats.

7,23), or D and E (Ref. 18)] display similar phosphorylation responses, peptide maps and immunological crossreactivity with the other stathmin spots 7.'e.~9,24.Each set is composed of up to three increasingly acidic, and presumably increasingly phosphorylated, spots 7.~3. Transfection experiments recently demonstrated z4 that, in uiuo, the same cDNA directs the synthesis of the 19 kDa forms of stathmin as well as of phosphoproteins '16' and '17', which shows that these two sets are also postor co-translational variants of the same protein. It is not clear at present whether they derive from the a and [3 isoforms or from other unphosphorylated isoform(s), as yet unidentified. The phosphorylation pattern of the various stathmin forms is dependent on the biological signal and on the resulting intracellular pathway(s) activated. Agents that activate the cAMP pathway (e.g. adenosine, forskolln) or mimic it, when applied alone, stimulate the phosphorylation only of the 19 kDa forms of stathmin 9.1°,13,19. On the other hand, growth factors, agents stimulating protein kinase C, Ca~" ionophores and other cell activators induce the incorporation of phosphate into the most basic spots (spot I) of the stathmin forms '16' and '17' as well 7.13.16,18,19,23. The combined action of both cAMPdependent and independent effectors results in a stronger phosphorylation of the 19 kDa forms than with either agent alone, and it also leads to the additional incorporation of phosphate into the more acidic forms (2 and 3) of '16' and ' 17'13'19. Phosphorylation of various sites on stathmin isoforms may therefore be interdependent, and possibly even sequential, with phosphorylation of one site favoring phosphorylation of another one in response to a different regulatory signal.

acids 2e, a feature that i~. characteristic of the consensus phosphorylation site for 'proline-directed' kinases2s.z9. One such protein kinase was indeed described in rat pheochromocytoma cells, and it is activated in PC12 cells by NGF28, which also stimulates phosphorylation of stathmin forms PI-P3, '16' and '17' in the same cells ~9. Molecularstructure afld pmperUes Stathmin was purified from rat and Interestingly, the cell-cycle-associated bovine brainZ2L it is an elongated 2~, 149- protein kinase p34ceczalso has the same amino acid protein n~ (calculated proline-directed phosphorylation-site molecular mass: 17269Da), which specificity29. The existence of several remains soluble after boiling2, possibly sites for distinct protein kinases is most because it contains many charged likely responsible for much of the residues. The cDNA-derived amino acid observed diversity of stathmin molsequence of the protein",~-2~ (Fig. 3) ecular forms. contains a proline-rich region followed by a 78 resk]ue predicted a-helical Stathmln expression:developmentaland domain with a 'heptad repeat' struc- functional roles The expression of stathmin is reguture~-, the first and fourth residues of each repeat being hydrophobic or lated by numerous factors, mostiy related uncharged residues, a feature known to to the proliferation and differentiation be able to yield coiled-coil interacting states of cells. Stathmin expression is structures. Although there is no exper- much higher in T lymphocytes stimuimental evidence yet, such structures lated for proliferation than in their restcould be responsible for interactions ing state ~7, and acute leukemic T cells between stathmin molecules or with express even more stathmin than norother cell components ,nvoived in the mal proliferating lymphoid cells s. In culbiological actions of stathmin. Except tured muscle cells, the expression of for a weak homology" with coiled-coil stathmin is highest in the proliferating regions of some intracellular matrix myoblasts and decreases to low levels proteins, no specific homologies with during their differentiation into funcsequences corresponding to known bio- tional myotubes ~5. Stathmin expression is also very high in various multipotenlogical properties were identified. Although stathmin contains no tyro- tial embryonic carcinoma cells24, its sine residue, it does contain eleven set- level being decreased alter the retinoic ine and two threonine residues, several acid and cAMP-induced endodermal difof which are located in consensus phos- ferentiation of F9 teratocarcinoma cells, phorylation-site sequences. Serines 16, as well as in their immortalized, differ46 and 63 are thus most likely potential entiated but still proliferating derivaphosphorylation sites for the cAMP- tives24. In the adult rat, stathmin is dependent protein kinasen2s, for which stathmin is a good substrate both in expressed at very different rates in difvitro ~z,z° and in vivo~Z'zL Serines 25 and ferent tissues, both at the protein and 38 are immediately followed by a pro- mRNA levels 3°. It is most abundant in line residue and then by basic amino brain 2,~2,3°.31, essentially because of its

Altogether, it appears that the pattern of stathmin forms and phosphorylation reflects the general state of activation of the cell, resulting from the combined action of multiple regulatory signals through diverse second messenger pathways.

303

TIBS 16 - AUGUST 1991 high expression in neurons ~. In the adult, these tissue-specific differences in stathrnin expression most likely reflect the diversity of the differentiated functions of mature cells and tissues. The high abundance of stathrnin in embryonic carcinoma cells suggests that it might also be biologically important at early stages of embryonic development 24. The expression of stathmin is much higher at the neonatal stage than in any adult tissue 3°. In brain, the levels of stathrnin protein and mRNA actually reach a peak around birth I~,22.31.Furthermore, whereas the range of stathrnin concentration is I to 200 in the adult rat, it is only 1 to 20 at the neonatal stage, brain being still the richest 3°. The high expression of stathrnin and its lower tissue dependence at the neonatal stage most likely reflects a feature common to developing tissues, namely the importance of the regulation of cell proliferation and differentiation. Its abundance in testis ~°,~, essentially in germ cells in their meiotic phase 32, further supports this interpretation.

Phylogeneticconservationand a gene family in addition to its ubiquity, stathrnin is also generally well-conserved throughout evolution27,3". Antibodies directed against a peptide located near the amino terminus of rat stathmin recognize a stathrnin-like 19 kDa molecule in vertebrate classes from fish to marnrnals3°. Furthermore, isoelectric variants similar to those of rat stathrnin were detected in all species tested, by both direct silver-staining~ and irnmunodetection:"j. Stathrnin is actually extremely well-conserved at least among mammals, since a single conservative difference exists between the cDNA-derived human and rat amino acid sequences '~6. Such high conservation suggests that stathrnin might have an essential and most likely general role in cellular regulatory processes. Although several stathmin rnRNAs of increasing size can be detected, they probably correspond to differences in the lengths of their 3'-untranslated sequences, as suggested by the isolation of several human cDNA clones that differ in the use of distinct polyadenylation sites 26:-'7.High-stringency Southern blot data also suggest the existence of a single gene for human stathmin ~'27. In agreement with these indications, a single gene for stathmin was recently localized on human chromosome 1 (Ref. 33).

304

t lowever, low-stringency Southern blot analysis suggests that other stathrain-related genes might exist. SCGIO is a neuron-specific, partially membranebound protein that accumulates in the growth cones and perinuclear cytoplasm of developing and regenerating neurons34. Its amino acid sequence~ is highly homologous (74%) to that of stathmin 25 (Fig. 3). In addition to its stathmin-like domain, SCGIO possesses a 32-amino acid amino-terminal domain that is probably responsible for its interaction with membranes 34. In its stathmin-like domain, the predicted ahelical region with its heptad organization is conserved, as are four out of the five predicted phosphorylation sites, indicating that SCGIO may also be phosphorylated in vivo. SCG10 and stathmin thus belong to a common gene family25,34 that shares most of the stathmin-sequence domain. Proteins like SCG10 are specifically expressed in given cell types and possess specific additional domains, possibly related to corresponding regulatory processes and cell functions. Stathmin, on the other hand, might represent the ubiquitous and generic functional domain of the protein family, its regulation and specific functions being related to the particular state of proliferation and differentiation of each cell type.

late the target biological responses? Perturbations of stathmin expression and function with transfection, antisense and antibody experiments are now crucial to answ~:r this question.

Acknowledgements 1 wish to thank all my collaborators for their help in preparing this manuscript and H. L. Cooper, S. Hanash and U. K. Schubart for communicating manuscripts before their publication.

References

1 Hunter, T. (1987) Cell 50, 823-829 2 Sobel, A., Boutterin, M-C., Beretta, L., Chneiweiss, H., Doye, V. and Peyro-Saint-Paul, H. (1989) J. Biol. Chem. 264, 3765-3772 3 Pasmantier, R., Danoff, A., Reischer, N. and Schubart, U. K. (1986) Endocrinology 19, 1229-1238 4 Braverman, R., Bhattacharya, B., Feuerstein, N. and Cooper, H. L. (1986) J. Biol. Chem. 261, 14342-14348 5 Hanash, S. M., Strahler, J. R., Kuick, R., Chu, E. H. Y. and Nichols, D. (1988) J. Biol. Chem. 263, 12813-12815 6 Hailat, N., Strahler, J., Melhem, R., Zhu, X. X., Brodeur, G., Seeger, R. C., Reynolds, C. P. and Hanash, S. (1990) Oncogene 5, 1615-1618 7 Peyron, J-F., Aussel, C., Ferrua, B., H~ring, H. and Fehlmann, M. (1989) Biochem. J. 258, 505-510 8 Gullberg, M., Noreus, K., Brattsand, G., Friedrich, B. and Shingler, V. (1990) J. BioL Chem. 265, 17499-17505 9 Beretta, L., Boutterin, M-C., Drouva, S. and Sobel, A. (1989) Endocrinology 125, 1358-1364 10 Beretta, L., Boutterin, Me. and Sobel, A. (1988) Endocrinology 122, 40-51 11 Sobel, A. and Tashjian, A,H., Jr (1983) J. Biol. Is stathmln a general reiay Integrating Chem. 258, 10312-10324 various signal transductlon pathways? 12 Chneiweiss, H., Beretta, L., Cordier, J,, Much has been learned during the Boutterin, M-C., Glowinski, J, and Sobel, A. (1989) J. Neurochem. 53, 856-863 last few years about this ubiquitous and highly conserved protein, about its gen- 13 Chneiweiss, H., Cordier, J., Doye, V., KopDel, J., Beretta, L. and Sobel, A. (1991) Soc. NeuroscL eral implication in developmental and Abstr. 16, 340 functional regulation, as well as about 14 Toutant, M. and Sobel, A. (1987) Dev. BioL 124, 370-378 its structure and the regulation of its A., Peyro-Saint-Paul, H., Koppel, J. and phosphorylation and expression. The 15 Sobel, Doye, V. (1989) EMBO Workshop on Cellular sensitivity of stathmin to a variety of and Molecular Biology of Muscle Development, Cambridge (UK), abstract P2 regulatory signals and the existence of specific patterns of phosphorylation in 16 Le Gouvello, S., Chneiweiss, H., Tarantino, N., Sobel, A. and Debre, P. (1990) Cell Biol. Int. response to certain pathways suggest Rep. 1[ 67 that this protein might play a general 17 Cooper, H. L., McDuffie, E. and Braverman, R. (1989) J. Immunol. 143, 956-963 ro~e as an intracellular relay for the transduction of multiple signals from 18 Cooper, H. L., Fuldner, R., McDuffle, E. and Braverman, R. (1990) J. ImmunoL 145, the cell's extracellular environment. 1205-1213 Furthermore, the complex interactions 19 Doye, V., Boutterin, M-C. and Sobel, A. (1990) J. BioL Chem. 265, 11650-11655 of such signals yielding characteristic L., Houdouin, F. and Sobel, A. (1989) stathmin phosphorylation patterns indi- 20 J.Beretta, Biol. Chem. 264, 9932-9938 cate that stathmin might integrate 21 Schubart, U. K., Alago, W., Jr and Danoff, A. (1987) J. Biol. Chem. 262, 11871-11877 diverse regulatory signals and path22 Doye, V., Soubrier, F., Bauw, G., Boutterin, M-C., ways. Beretta, L., Koppel, J., Vandekerckhove, J. and The challenge for the future is to Sobel, A. (1989) J. BioL Chem. 264, ~mderstand the precise function and 12134-12137 mode of action of stathmin at the 23 Mary, D., Peyron, J. F., Auberger, P., Aussel, C. and Fehlmann, M. (1989) J. Biol. Chem. 264, rnoiecular level. How are the integrated 14498-14502 signals relayed to regulate and/or rnodu- 24 Doye, V., Kellermann, 0., Richoux, V., Renard,

TIBS 16 - AUGUST1991 J. P., Buc, M. H. and Sobel, A. (1990) J. Ce// BioL 111, 482a 25 Schubart, U. K., Das Banerjee, M. and Eng, J. (1989) DNA 8, 389-398 26 Maucuer, A., Doye, V. and Sobel, A. (1990) FEBS Lett. 264, 275-278 27 Zhu, X.X., Kozarsky, K., Strahler, J. R., Eckerson, C., Lottspeich, F., Melhem, R., Lowe, J., Fox, D. A., Hanash, S. M. and

THE PARASITIC FP,OTOZOA Trypanosoma and Leishmania cause numerous diseases in both man and domestic animals. Transmitted by blood-sucking insects, they infect the circulation, the lymphatics and other organs such as the brain and the heart. In humans, Trypanosoma cruzi is the agent of the often-fatal South American Chagas disease, for which there is no effective chemotherapy. African sleeping sickness is found in many regions of Africa, caused by either Z brucei gambiense or Z brucei rhodesiense, while in cattle, Z congolense causes the corresponding disease known as nagana. Leishmania are widely spread in nature, causing a variety of diseases, including human visceral leishmaniasis (kala-azar, Leishmania donovani), human cutaneous leishmaniasis (oriental sore, L tropica) and mucocutaneous leishmaniasis (espundia, L. braziliensis) I. The drugs used to treat these diseases are often fairly toxic to the host themselves. These include the organic arsenicals (melarsoprol), pentavalent antimonials and suramin, which was first used in 1920 (Ref. 2). The only exception appears to be the recent introduction of difluoromethyiornithine (DFMO), although this was originally developed as a potential anticancer agent and requires doses in excess of 20 g a da~,. In general, the majority of drugs in use have been shown to be carcinogens, to damage vision, or to have general toxic effects in humans. The limited success and liability of these treatments has led to research on the basic metabolic processes of the parasites; the evaluation of significant distinctions between the biochemistry of the host and parasite will hopefully lead to the development of logical approaches to chemotherapy. This has ¢. Walsh, M. Bradleyand K. Nadeau are at the Departmentof BiologicalChemistryand Molecular Pharmacology,HarvardMedical School, Boston,MA02115, USA.

Atweh, G. F. (1989) J. Biol. Chem. 264, 14556-14560 28 Vulliet, R., Hall, F. L., Mitchell, J. P. and Hardie, D. G. (1989) J. BioL Chem. 264, 16292-16298 29 Moreno, S. and Nurse, P. (1990) Cell 61, 549-551 30 Koppel, J., 6outterin, M-C., Doye, V., PeyroSaint-Paul, H. and Sobel, A. (1990) J. Biol. Chem. 265, 3703-3707

31 Schubart, U.K. (1988) J. Biol. Chem. 263,

12156-12160 32 Amat, J. A., Fields, K. L. and Schubart, U. K. (1990) Mol. Reprod. Dev. 26, 383-390 33 Ferrari, A. C., Seuanez, H. N., Hanash, S. M. and Atweh, G. F. (1990) Genes Chromosomes Cancer 2, 129-129 34 Stein, R., Mori, N., Matthews, K., Lo, L. C. and Anderson, D. J. (1988) Neuron 1, 463-476

Molecular studies on trypanothione reductase,atarget for antiparasiticdrugs

,

Trypanosoma and Leishmania are parasitic protozoa that cause a variety of diseases, which include African sleeping sickness and oriental sore. Attempts to determine pharmaceutically exploitable differences between host and parasite biochemistry have identified the unique trypanothione pathway as a possible target. This pathway includes the enzyme trypanothione reductase, the parasite analogue of glutathione reductase.

shown that there are major and potentially exploitable differences between host and parasite biochemistry, including thiol and polyamine metabolism 3. It is this aspect of the biochemical pathways in Trypanosoma and Leishmania, in particular the close interaction of the glutathione pathway with polyamine biosynthesis, that will be discussed here. Polyaminesand glutathione The polyamines (putrescine, spermidine and spermine) and glutathione (7"Lglutamyl-L-cysteinylglycine) are found in millimolar concentrations in most biological systems and are components of many important biological processes. These include cell growth and differentiation and regulation of important enzymatic processes. Glutathione, for example, plays a pivotal role in the management of oxidative stress and in the maintenance and regulation of intracellular thiol/disulfide redox balance. It serves as a cofactor for peroxide reductions, ribonucleotide reduction, cis/ trans isomerizations, and, in higher organisms, serves in the conjugation and detoxification of foreign substances 4. The levels of reduced glu-

© 1991,Elsevier Science Publishers, (UK) 0376-5067191/$02.00

tathione are maintained by the reduction of oxidized glutathione by the NADPH-dependent enzyme glutathione reductase. Polyamines are required for optimal growth in many cell types, but their precise regulatory functions remain obscure 5.

Oxidative stress During their infective cycle, Trypanosoma must survive the rigors of not only the host's oxidative phagocytic macrophages, but also the oxygen intermediates generated during normal metabolism. The Trypanosorna and Leishmania are deficient in some of the enzymes that form an important defense against oxygen toxicity in most organisms. For example, they lack the typical catalase/peroxidase hemoproteins 6.7, although some appear to contain a thiol-dependent peroxidase ~. These observations, along with the known sensitivity of Trypanosoma to oxidative stress 9, suggested that these parasites would depend heavily on the coupled glutathione peroxidase/reductase activities to detoxify these potentially lethal metabolites. That this system can be easily overloaded in Trypanosoma is consistent with their

305