No longer an exclusive club: eukaryotic signalling domains in bacteria

reviews

No longer an exclusive club: eukaryotic signalling domains in bacteria Christopher J. Bakal and Julian E. Davies

Reversible phosphorylation of serine, threonine and tyrosine residues by the interplay of protein kinases and phosphatases plays a key role in regulating many different cellular processes in eukaryotic organisms. A diversity of control mechanisms exists to influence the activity of these enzymes and choreograph the correct concert of protein modifications to achieve distinct biological responses. Such enzymes and their adaptor molecules were long thought to be specific to eukaryotic cellular processes. However, there is increasing evidence that many prokaryotes achieve regulation of key components of cellular function through similar mechanisms.

Christopher J. Bakal is at the Ontario Cancer Institute, Princess Margaret Hospital, 610 University Avenue, Toronto, Ontario, Canada M5G 2M9; and Julian E. Davies is in the Dept of Microbiology and Immunology, The University of British Columbia, 6174 University Blvd, Vancouver, BC, Canada V6T 1Z3. E-mail: jed@ interchange.ubc.ca

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The roles that phosphorylation and dephosphorylation play in the regulation of prokaryotic protein activity became apparent in the late 1970s. Early studies, however, focused primarily on the phosphorylation of histidine and aspartic acid, and bacteria were assumed not to possess the serine/threonine and tyrosine kinases of the type described by Hanks1. However, since the early 1990s, studies based on a combination of the polymerase chain reaction (PCR), X-ray crystallography and genomic sequencing have shown that prokaryotes possess many of the features of eukaryotic enzymatic signalling machinery. The milestones in the relatively new field of prokaryotic signalling are listed in Box 1. In the past nine years, it has been revealed that prokaryotes such as Streptomyces sp., Cyanobacteria and Myxococcus xanthus seem to have evolved complex signal-transduction networks that have fashioned their ability to differentiate and live in multicellular communities. Serine/threonine kinases: few known targets Although early biochemical studies had suggested the presence of serine/threonine and tyrosine protein

kinases in Escherichia coli2,3, it was not until 1991 that a gene, pkn1, encoding a Hanks-type serine/ threonine protein kinase (STPK) was cloned from the bacterium Myxococcus xanthus4. When pkn1 was expressed in Escherichia coli grown in the presence of radiolabelled Pi, phosphorylation of Pkn1 on both serine and threonine was observed, indicating that phosphate was introduced via an autophosphorylation event. This autophosphorylation suggested that Pkn1 might also phosphorylate exogenous proteins (although certain proteins, such as nucleoside diphosphate kinase, that are not protein kinases can undergo autophosphorylation). Northern blot analysis indicated that expression of pkn1 is developmentally regulated, and a pkn1 knockout in Myxococcus xanthus resulted in premature differentiation and spore formation. Similar observations followed in further studies with Myxococcus xanthus5–7, as well as the eubacteria Anabaena PCC71207–9 and Streptomyces coelicolor10–13. One or more STPKs were cloned, expressed in Escherichia coli, found to autophosphorylate serine and threonine residues and analysed for developmental/ morphological defects by gene disruption in the native host. These results are summarized in Table 1. Interestingly, the phenotypes of many of the STPK knockouts are relatively weak – typically, STPK gene disruptions will cause a reduction in, for example, aerial mycelium or spore formation but rarely result in a complete developmental block. This suggests that the eubacteria possess protein kinases with partially redundant functions, a phenomenon found commonly in eukaryotes. It should also be emphasized that laboratory studies do not simulate the actual habitat of these bacteria, where the protein kinases might play different, and possibly more crucial, roles in maintaining bacterial community structure. Although the identifications and subsequent gene disruptions established the role of STPKs in prokaryotic development, many of these studies were incomplete in that endogenous substrate, upstream activators or the targets and effects on gene regulation were unidentified. That prokaryotic STPKs have direct roles in transcriptional activation was shown in studies of Pkn9 from Myxococcus xanthus. Entire deletion of the pkn9 gene, which encodes a transmembrane STPK, resulted in severe reduction in cell progression through development and spore formation, and reduced expression of five membrane proteins (called KREP9 proteins)6. This was the first demonstration of the importance of a transmembrane STPK in modulating transcriptional events and implies that Pkn9 might serve as a receptor for extracellular signals. Interestingly, when only the kinase domain of the protein was disrupted, the KREP9 proteins remained unexpressed, but spore formation was normal and the impact on development was not as severe. This suggests that Pkn9 interacts with other signalling proteins that are crucial to the development of the organism. A well-characterized, although far from completely understood, pathway involving STPK signalling is the phosphorylation of the AfsR regulatory protein by the phosphokinase AfsK in

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trends in CELL BIOLOGY (Vol. 10) January 2000

reviews BOX 1 – MILESTONES IN ELUCIDATION OF PROKARYOTIC SIGNALLING 1958: Discovery that glycogen phosphorylase activity can be regulated by phosphorylation on serine. The field of signal transduction begins. 1986: Src-homology domains SH2 and SH3 are discovered. 1988: Biochemical studies show eukaryotic-like kinase activity in Escherichia coli. 1989: Discovery of eukaryotic-like protein-serine/threonine phosphatases encoded by the genome of the bacteriophages l and f80. 1990: SH2 domains bind pTyr proteins. Yersinia pseudotuberculosis virulence plasmid encodes an essential protein-tyrosine phosphatase (PTP). 1991: pkn1 encoding a eukaryotic-like serine/threonine kinase from Myxococcus xanthus is cloned. 1992: A membrane protein from Pseudomonas solanacearum is shown to be phosphorylated on tyrosine. 1993: pknA from Anabaena sp. strain PCC7120 is cloned. A gene encoding a PTP, iphP, from Nostoc commune is cloned. 1994: AfsR is regulated by the serine/threonine protein kinase (STPK) AfsK in Streptomyces coelicolor. AfsK is capable of autophosphorylation on tyrosine. Numerous proteins phosphorylated on tyrosine are detected in Streptomyces sp. 1995: Pkn2 from Myxococcus xanthus phosphorylates β-lactamase. 1996: ptpA, which encodes a PTP, is cloned from Streptomyces coelicolor. 1997: The structure of an enzyme required for aminoglycoside antibiotic resistance reveals homology to eukaryotic protein kinases. PDZ domains are found in bacterial proteins. 1998: The Mycobacterium tuberculosis genome is completely sequenced and 13 eukaryotic-like kinases are revealed. Synechocystis genome sequencing reveals numerous eukaryotic-like signalling proteins. 1999: Structure of CheA shows that the regulatory domain resembles two SH3 domains. SH3 domains are found in bacterial proteins. Ap-ATPase domains, TIR domains and a caspase are discovered in Streptomyces coelicolor.

Streptomyces coelicolor10. AfsK has a similar catalytic core to eukaryotic STPKs, and, although it does not appear to possess any distinct membrane-spanning or hydrophobic regions, it is localized to the membrane fraction. Furthermore, the recombinant protein is capable of phosphorylating serine and threonine residues of a phosphoprotein from Streptomyces coelicolor, AfsR, which is responsible for transcriptional stimulation of genes involved in antibiotic biosynthesis14. AfsK and Pkn2 from Myxococcus xanthus (in which b-lactamase was identified as a potential substrate5) are two of the very few examples in which a Hanks-type protein kinase has been demonstrated to phosphorylate an exogenous protein in a prokaryote. The two cases differ in that AfsK phosphorylates a protein known to be regulated by phosphorylation and implicated in cellular regulation in Streptomyces. Whether b-lactamase is regulated by phosphorylation in Myxococcus xanthus in the environment is still unknown. Additional studies have confused the true role of AfsK; crude cell extracts of Streptomyces coelicolor were capable of phosphorylating AfsR at a much faster rate than purified AfsK, and disrupting the afsk gene reduced antibiotic production significantly, with AfsR still phosphorylated to the same extent as the wild type10. Recent studies with Streptomyces griseus have expanded on the role(s) of the AfsK–AfsR interaction and indicate that additional STPKs might be involved59. It appears that many streptomycetes have the AfsK–AfsR regulatory system. Bacterial Ras family members: similar form and function In eukaryotes, activation of many signalling pathways involves the recruitment of kinases to membrane-associated signalling complexes, where the GTP binding of proteins such as the proto-oncogene Ras serves to initiate the signalling cascades. Ras-like molecules have been found in Pseudomonas aeruginosa15 and Mycobacterium tuberculosis16, and both are regulated by GTP binding, although the involvement

of these proteins in signal transduction is less clear. Myxococcus xanthus also possesses a protein, MglA, that shares identity with low-molecular-mass GTPases, such as Ras, Rab, Rho and Sar1p, that are required for multicellular development and gliding motility17. When integrated into the Myxococcus

TABLE 1 – PHENOTYPES OF STPK KNOCKOUTS Organism

STPK

Disruption phenotype

Myxococcus xanthus

Pkn1 Pkn2 Pkn5 Pkn6 Pkn9

Streptomyces sp.

AfsK Pkg2 PknA PknD

Premature differentiation and spore development 30%–50% reduction in myxospores, faster development Forms fruiting bodies faster Slower development Altered fruiting body formation, absence of KREP9 proteins in membrane, reduced spore production Antibiotic production reduced significantly Disintegrating aerial hyphae Smaller cell size, less heterocysts under combined N2-limiting conditions Growth rate 20% of wild-type under N2-fixing conditions, enhanced PII phosphorylation Under N2-fixing conditions: stagnated growth after 4–5 days. Aberrant heterocyst structure. 10.6% of wild-type nitrogenase activity.

Anabaena sp. PCC7120

PknE

Abbreviation: STPK, serine/threonine protein kinase.

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reviews xanthus genome, the SAR1 gene complements the sporulation defect of a DmglA strain18. Furthermore, a second-site mutation in combination with SAR1 complements the defect in motility as well. This experiment provides compelling evidence that, in bacteria, as in eukaryotes, GTPases play central roles in signal transduction. Tyrosine kinases: raiders of the lost src Nearly a decade of studies into prokaryotic signalling has clearly shown STPKs are used extensively by bacteria. However, information regarding prokaryotic tyrosine phosphorylation has not accumulated as quickly. Phosphotyrosine-containing proteins have been identified in a wide variety of prokaryotes by PY-specific antibody crossreactivity in cell extracts and kinase assays19. So far, Streptomyces sp. seem to use this posttranslational modification to the greatest extent as numerous proteins have been observed to be phosphorylated on tyrosine20, whereas, in some organisms only one or two proteins are so modified. In all cases, however, the appearance and quantity of phosphotyrosine-containing proteins is dependent on changes in growth (logarithmic to stationary), media20 and environmental conditions (the presence of fixed nitrogen21 or light22). A compelling example of factors that play a role in tyrosine phosphorylation was observed recently in Streptomyces griseus, where cAMP affects protein tyrosine phosphorylation23. Modulation of a protein tyrosine kinase by cAMP has also been reported in Acinetobacter calcoaceticus24 (interestingly, the regulatory effect of cAMP is observed exclusively with STPKs in eukaryotes25). Two questions arise immediately from these preliminary data: what are these tyrosine-phosphorylated proteins? And what kinases are responsible for their phosphorylation? Few bacterial proteins shown to be modified by phosphorylation on tyrosine have been identified. Those that have, such as TypA from Escherichia coli26 and flagellins a and b from Pseudomonas aeruginosa27, would appear to be examples of phosphorylation at the end of potential signalling cascades, and not enzymes that are activated by phosphorylation sequentially, as is typical of eukaryotic signal transduction. AfsK can autophosphorylate on serine and tyrosine in vitro, but it is not known whether this occurs in vivo and whether AfsK acts as a tyrosine kinase for other substrates. Autophosphorylation of tyrosine has also been reported recently for the Wzc protein from Escherichia coli by Vincent et al.28 The significance of this finding is not yet apparent, and Wzc has not been implicated in a signal-transduction cascade. The authors speculated that Wzc is a tyrosine kinase involved in pathogenicity, but no supporting evidence was provided. Sequence information has not yet revealed whether homologues of mammalian receptor-type tyrosine kinases [such as the epidermal growth factor (EGF) receptor kinase] and src-family kinases are to be found in bacteria. This is perhaps not surprising given the fact that genomic sequencing of plants, yeast and fungi has not revealed such types of kinases. It is

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possible that bacteria possess tyrosine kinases that are indistinguishable by sequence from STPKs. Thus, many of the putative kinases identified by prokaryotic sequence gazing29 could in fact be tyrosine kinases. Phosphatases: the yin and yang of bacterial signalling Regulation of signalling pathways involves coordinated action of not only the kinases but also associated protein phosphatases. In eukaryotic signal transduction, this role is carried out by the PPP and PPM family of protein-serine phosphatases, and the low-molecular-weight (LMW) and conventional families of protein-tyrosine phosphatases (PTPs). Prokaryotic homologues of these enzymes have been found29,30, but, as with the STPKs, the role that many of these phosphatases might play in bacterial signalling is not clear. There is evidence that the PPPs PrpA and PrpB from Escherichia coli are involved in signal transduction that senses protein misfolding caused by heat shock or other stress31. Regulation of prokaryotic protein kinases by PPPs was suggested by studies of Anabaena sp. PCC7120, in which PrpA is a protein serine/threonine phosphatase, which is closely linked genetically to the pknE gene, encoding a STPK9. Disruption of either gene affects similar biological processes (heterocyst structure development, cell growth under nitrogen-fixing conditions and nitrogen-fixing activity), suggesting that they affect similar targets. Interestingly, prpA appears to be expressed constitutively, whereas pknE is regulated during the process of heterocyst development, suggesting that the equilibrium between phosphorylation and dephosphorylation is regulated at the level of kinase expression. The role of the PPM family SpoIIE in sporulation, and possibly septation, has been elucidated in a series of studies30. RsbX and RsbU, two other PPMs from Bacillus subtilis, participate in regulatory pathways that result in the transcriptional activation of stress-response genes32. Conventional PTPs do exist in prokaryotes. Many of these enzymes are virulent elements secreted by pathogenic bacteria and target the signalling pathways of eukaryotic hosts. Most likely, these genes have been acquired from mammalian host organisms via horizontal gene-transfer events. PTPs that probably have bacterial ancestry have been discovered, such as IphH from the cyanobacterium Nostoc commune UTEX58421, and have been shown in vitro to possess protein phosphatase activity33. However, the physiological substrates and potential activators remain unknown. LMW PTPs, such as PtpA from Streptomyces coelicolor A3(2)34 and Ptp from Acinetobacter johnsonii35, have also been characterized to have in vitro activity. Overexpression of the ptpA gene in Streptomyces lividans led to an increase in the production of antibiotics and A-factor36. However, disruption of the ptpA gene in Streptomyces coelicolor A3(2) had no detectable effect on similar metabolite production or mycelium or spore formation34. The study of PtpA implies, again, that redundancy might be a key feature of both eukaryotic and bacterial signal transduction. trends in CELL BIOLOGY (Vol. 10) January 2000

reviews ‘Eukaryote-specific’ signalling domains In eukaryotic organisms, the formation of complex networks of interacting proteins is facilitated by conserved protein modules or domains that regulate signal transduction by mediating protein–protein interactions. Many of the proteins possess multiple domains that facilitate the simultaneous association of two or more binding partners (reviewed in Ref. 37). FHA (Forkhead Associated) domains mediate protein–protein binding that is dependent on phosphorylated serine or threonine and are found in eukaryotic proteins that are involved in cell-cycle control38. FHA domains are found in numerous known and putative prokaryotic transcriptional regulators such as FraH from Anabaena sp*. Interestingly, this domain is also found in an ABC transporter from Synechocystis, and in an open-reading frame on the reverse strand of a STPK from Streptomyces coelicolor39. As these domains seem to be present in both membrane and possibly DNA-binding proteins, prokaryotes could also use FHA domains in signal-transduction pathways that could involve transcriptional activation resulting from signalling events on the membrane. Interestingly, FHA domains are found in those bacteria that also have Pkn2-type kinases and PP2C-type phosphatases. Could the FHA domains/Pkn2/PP2C proteins represent a conserved pathway40? PDZ domains (for domain present in PSD-95, Dlg and ZO-1/2) have been observed in more than 60 eukaryotic cytosolic proteins, many of which are located at specific regions of cell–cell contact, such as tight junctions, septate junctions and synaptic junctions. Many proteins containing one or more PDZ domains have been implicated in localizing membrane proteins to specific subcellular regions by interacting directly with the C-terminal amino acid residues of transmembrane proteins41, such as with the E(S/T)DV motif at the C-terminus of certain ionchannel subunits42. In higher eukaryotes, these interactions promote clustering of voltage-gated and ligand-gated ion channels at synapses43. A number of proteins containing PDZ domains exist in prokaryotes44, including HtrA-like serine proteases and S2P-like metalloproteases. Evidence indicates that the C-terminal polypeptide-binding function of these domains might be common to all three divisions of life and, by inference, might have been present in the last common ancestors. As with other eukaryotic-like domains found in bacteria, many of the PDZ-containing proteins are secreted by pathogenic organisms. However, the participation of PDZ domains in regulating bacterial signalling pathways is not well established. One of the proteins, SpoIVB of Bacillus subtilis, contains a PDZ signalling module; this 427-residue protein, which contains a single PDZ domain, is an important component of the intercompartmental signal-transduction pathway that activates the sigma factor s-k in the mother cell of the sporulating complex45. As SpoIVB interacts with several proteins of the forespore membrane, the SpoIVB PDZ domain could be involved in the formation of a multiprotein structure that engages s-k processing in the motherspore and trends in CELL BIOLOGY (Vol. 10) January 2000

SH3 CheA Chemotaxis, motility

Pkn9 STPK

STPK

PDZ SpoIVB

AfsK

Sporulation

MgIA AfsR

Ras-GTPase

PS PT TIR

Ap-ATPase

Pk3 STPK CASP

STPK Pkn1 Development, sporulation

Antibiotic production PTP

Apoptosis?

PtpA

trends in Cell Biology

FIGURE 1 The roles of eukaryotic-like signalling domains in bacterial signal transduction. Pkn1, Pkn9 and MglA are from Myxococcus xanthus; AfsK, AfsR, PtpA and Pk3 from Streptomyces coelicolor; SpoIVB from Bacillus subtilis; and CheA from Thermotoga maritima. Abbreviations: CASP, caspase; PS, phosphorylated serine; PT, phosphorylated threonine; SH3, Src-homology 3; STPK, serine/threonine protein kinase.

mediates signalling events within the forespore. The ligand for the SpoIVB PDZ domain is not known, and none of the proteins thought to interact with SpoIVB has the S/TXV-COOH motif. However, some PDZ domains can bind to internal peptides; therefore, SpoIVB could be binding to other regulatory proteins in this manner46. The case of SpoIVB, and the fact that, for example, a putative integral membrane protein from Streptomyces coelicolor (ID:CAB41567) possesses a PDZ domain, implies that, in bacteria, as in eukaryotes, PDZ domains might also be involved in signalling events initiated at the membrane (Fig. 1). Src-homology 3 (SH3) domains bind proline-rich peptide sequences containing the consensus PXXP that forms a left-handed polyproline type II helix47. In eukaryotic organisms, the principal role of SH3 domains is the formation of functional oligomeric complexes at defined subcellular sites, frequently in conjunction with other modular domains37. Two different approaches have been used to demonstrate the presence of SH3 domains in prokaryotic organisms. By sequence gazing, several bacterial proteins and open-reading frames containing homologues of these domains have been detected48,49. A subset of bacterial proteins from Bacillus subtilis and Staphylococcus simulans that contain the SH3 domains also contain lytic enzymatic domains. These lysins are predicted to be involved in pathogen–host interaction or alternatively in cell division. The ‘pathogenic’ nature of some of these proteins suggests that SH3 domains were obtained in horizontal transfer events, similar to the SH2 domains found in Yersinia pseudotuberculosis50. Another set of bacterial SH3 domains have been found in known prokaryotic signalling proteins, which suggests an ancient (prokaryotic) origin for the SH3 domain. Synechocystis PCC6803 protein

*Note added in proof: Recently, the phosphodependent peptide binding capability of both eukaryotic and prokaryotic FHA domains was demonstrated [Durocher, D. et al. (1999) Mol. Cell 4, 387–394], implying that prokayrotic FHA domains are likely to be involved in signalling mechanisms similar to those of their eukaryotic counterparts.

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reviews sll0776 (gi 1006577) contains a C-terminal SH3 domain preceded by a Pkn2-type40 protein kinase domain, which resembles that of the previously described Pkn2 from Myxococcus xanthus. The function of this potential kinase is not known, but, since sll0776 has a putative transmembrane region, it might serve as a receptor. Further evidence that some bacterial SH3 domains have true ancestral origins, and that they are not the product of horizontal gene transfer, has come from structural studies of CheA, a signal-transducing histidine kinase51 from Thermotoga maritima that is a component of a signal-transduction pathway mediated by histidine phosphorylation. This pathway is similar to other ‘two-component systems’ or ‘phosphorelays’ (reviewed in Ref. 52). These pathways play a central role in regulating a wide variety of cellular responses, including bacterial chemotaxis, osmoregulation and sporulation. The signalling system is generally made up of two (or more) multidomain proteins. In the case of chemotactic signalling in Thermotoga maritima, transmembrane chemoreceptors regulate the histidine phosphorylation of CheA through the adaptor CheW, and the response regulator CheY obtains the phosphate from CheA and itself becomes a signal to change flagellar rotation. The regulatory domain that mediates the interaction between CheA and CheW, and thus kinase activity, structurally resembles two SH3 domains51. This domain, termed P5, is very different in amino acid sequence from the SH3 domains described previously. The P5 domain is present in other multidomain proteins in this cascade, namely the response regulator CheV and the adaptor protein CheW51. The presence of such distantly related SH3 domains in bacteria further substantiates the notion that they are distributed more widely in living organisms than was thought previously and raises the possibility that additional and novel SH3 domains are yet to be found. Signalling modules and bacterial apoptosis The ability to undergo apoptosis is a fundamental and crucial property of animal cells. Extracellular apoptotic stimuli result in the activation of a class of proteases termed caspases53; in the apoptotic response, caspases are activated by an amplifying proteolytic cascade54. The activated caspases then cleave specific proteins in the cell, leading to rapid cell death. Some bacteria have been known to undergo a process resembling apoptosis as a means of regulating cell density during the stationary phase and in response to viral infection. Rhizobium melloti undergoes a similar process during the development of bacteroids55. Do bacteria possess components of the apoptotic machinery? If so, do they function in bacterial apoptosis? Aravind et al. found that copies of the Ap-ATPase domain, a domain specific to proteins involved in apoptosis, are encoded by members of the actinomycetes and by Bacillus subtilis56. The bacterial Ap-ATPase domains more closely resemble those from the human apoptotic protease-activating factor APAF-1 than CED-4 from Caenorhabditis elegans. Given the functional similarity

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between CED-4 and APAF-1, this suggests that prokaryotic Ap-ATPase domains will also share similar mechanisms of action. Interestingly, AfsR from Streptomyces coelicolor contains not only the Ap-ATPase domain but also a TIR domain56, which is associated with protein–protein interactions in metazoan apoptotic molecules. As described earlier, AfsR acts downstream of the AfsK STPK and is necessary for activation of the genes responsible for biosynthesis of the antibiotic actinorhodin. As AfsR possesses two domains that have been shown to be involved in protein–protein interactions, this raises the possibility that AfsR has another important role beyond production of secondary metabolites – namely, bacterial apoptosis. Perhaps a more convincing argument for prokaryotic programmed cell death is the finding of caspase homologues in Streptomyces coelicolor and Rhizobium sp.56, indicating that caspases belong to an ancient and diverse protein superfamily. However, whether these play a role in prokaryotic apoptosis remains to be determined. Interestingly, the caspase protein from Streptomyces coelicolor also contains a eukaryotic-like kinase domain. As caspases are regulated by kinases57, and vice versa58, in metazoan apoptosis, the fact that the Streptomyces caspase is also the putative STPK Pk3 might indicate that these two types of regulation have been intimately related since the origin of the bacterial cell. Conclusions and future challenges Since the discovery of pkn1 eight years ago, the identification of a wide range of ‘eukaryotic-like’ signalling molecules in prokaryotes has ended the controversy about the origins of these enzymes and their related domains. The finding that these proteins have ancient origins further underscores their importance to all organisms and demonstrates the remarkable degree of physiological complexity possessed by many prokaryotes. The number of STPKs to be found in Streptomyces coelicolor alone could be significant, and many more ‘eukaryote-specific’ signalling domains (SH2, PH, PTB/PI, SAM, etc.) will probably be found in bacteria. There are likely to be tens of thousands of bacterial genera with many species that possess complex developmental cycles and participate in intercellular messaging; the discovery of STPK, SH3, PDZ, FHA and other signalling domains in the relatively very small number of bacterial genomes so far sequenced promises that many more such signalling domains will be found, emphasizing the ancient origins of these functions. Furthermore, there are often vast differences in the genomes and proteomes of seemingly related organisms. For example, the completed genome of Synechocystis PCC6803 is often seen as representative of all cyanobacteria. However, the cyanobacteria are an enormous clade, with many unique species. Calothrix sp. of the cyanobacterial lineage have genomes that are larger than 15 Mb in size (nearly five times the size of Synechocystis PCC6803), and nothing is known of others. Thus, we predict that there will be many more surprises to come, especially since it is estimated that more than 99% of trends in CELL BIOLOGY (Vol. 10) January 2000

reviews all bacteria have yet to be identified. Many challenges remain; most studies have concentrated on the identification of single proteins, and not on the pathways in which they participate. For example, although a large number of STPKs have been identified in microorganisms, the true substrates for these enzymes are unknown, apart from AfsK, for which one potential target is known. Furthermore, information regarding the regulation of the STPKs, such as potential receptors and/or the ligands themselves, is non-existent. A striking observation regarding both the characterized and putative STPKs of prokaryotes found thus far is that, unlike eukaryotic STPKs, the majority appear to be transmembrane proteins. For example, of the 13 known STPKs in Mycobacterium tuberculosis, nine are transmembrane and appear to have an extracellular ligandbinding domain. Of the seven found in Synechocystis sp. PCC6803, four appear to be transmembrane. Are these prokaryotic receptors/sensors? Lastly, the genes and specific physiological outcomes that are affected by activation or inactivation of these pathways are also generally unknown. Prokaryotic signalling has so far focused on the words of signal transduction, with the sentences, and ultimately the language, of signalling not yet within our grasp. Increasing knowledge of bacterial genomes will provide an appropriate dictionary that will be of significance in interpreting the evolution of posttranslational signalling systems in higher organisms. We predict that the future lies in studies of prokaryotic-like kinases and phosphatases in eukaryotes, and not the converse. This is as it should be in an evolutionary sense. References 1

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Acknowledgements The authors thank Chris Ponting for critical review of the manuscript, and Dusica Vujakilija for her assistance and generosity. This research was funded by the Natural Sciences and Engineering Research Council of Canada.

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Enigmatic annexins Annette Draeger *50th Harden Conference, Annexins, Wye College, Kent, UK; 1–5 September 1999. Organized by Stephen Moss.

Annette Draeger is in the Dept of Cell Biology, Institute of Anatomy, University of Bern, Switzerland. E-mail: draeger@ ana.unibe.ch

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Twenty years of intensive research into the annexins have furnished us with a wealth of information with respect to the structural characteristics and biochemical properties of these membrane-binding proteins. However, they have yet to be assigned a definitive function. It was thus only fitting that the Biochemical Society should choose ‘The Annexins’ as the subject of its 50th Harden Conference*. During the course of this unique international symposium – the first of its kind on annexins – many of the jumbled puzzle pieces gradually started to fit together. The meeting also marked the end of a three-year funding period of research on the structure and biological role of annexins by the European Union’s Fourth Framework Programme in Biotechnology (coordinator: A. LewitBentley, LURE, Orsay, France). Annexin structure Annexins are characterized by their ability to bind to anionic lipids in a Ca21-dependent manner and by their common structure: a stretch of about 70 amino acids repeated either four or eight times. However, even significant differences in amino acid sequence do

not necessarily lead to variations in tertiary structure. X-ray analyses of the annexin II-p11 and annexin I-S100C complexes have revealed striking resemblances between the two, which point to functional similarities in their interaction with their binding partners (A. Lewit-Bentley). Interestingly, N-terminal mutations in the annexin III protein have been shown to alter its membrane-binding properties in a manner that is reminiscent of a phosphorylation event (F. RussoMarie, ICGM, Paris). Site-directed spin-labelling studies of annexin XII (H. Haigler, Irvine, USA) have taken the X-ray-crystallographic approach even further and have furnished evidence of a novel transmembrane form1 with pH-dependent refolding and insertion into the lipid bilayer. Annexins as membrane ‘organizers’ The general organization of membranes depends critically upon the self-association of annexins into multimers, as demonstrated by electron- and atomic-force microscopy (A. Brisson, Groningen, The Netherlands), this event being realized largely by virtue of

0962-8924/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0962-8924(99)01683-9

the lipid-binding properties of these molecules. The self-organization of annexin V trimers into ordered arrays spanning the lipid surface might well provide a structural basis for the affiliation of other related – or unrelated – proteins. Evidence for annexin-induced modifications of membrane properties has come from G. Nelsestuen (St Paul, USA). Even when present at low densities at membrane surfaces, these molecules appear to be capable of influencing the association or dissociation of other membrane proteins. Thus, the overall regulation of cell membrane characteristics and signalling properties might be dependent on, or strongly influenced by, the presence and correct spatial organization of annexin molecules (see Fig. 1). The bilayer of which cell membranes are comprised contains cholesterolenriched lipid microdomains. These ‘rafts’, which participate in the formation of caveolae, have been implicated in transcytotic pathways within epithelial cells2. In vitro studies have revealed annexin XIII to be involved in the attachment and fusing of the trans-Golgi network and in its co-clustering with raft proteins (F. Lafont, Heidelberg, Germany,) thus making it a candidate for the organization of the raft structure itself – recruiting proteins to these clusters or, once recruited, influencing their half-life.

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