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105 Li, L. et al. (2001) A TSG101/MDM2 regulatory loop modulates MDM2 degradation and MDM2/p53 feedback control. Proc. Natl. Acad. Sci. U. S. A. 98, 1619–1624 106 Oh, H. et al. (2002) Negative regulation of cell growth and differentiation by TSG101 through association with p21Cip1/WAF1. Proc. Natl. Acad. Sci. U. S. A. 99, 5430–5435 107 Krempler, A. et al. (2002) Targeted deletion of the Tsg101 gene results in cell cycle arrest at

G1/S and p53 independent cell death. J. Biol. Chem. 29, 29 108 Hyatt, A.D. et al. (1991) Localization of the non-structural protein NS3 in bluetongue virus- infected cells. J. Gen. Virol. 72, 2263–2267 109 Fraile-Ramos, A. et al. (2002) Localization of HCMV UL33 and US27 in endocytic compartments and viral membranes. Traffic 3, 218–232 110 Ruland, J. et al. (2001) p53 Accumulation, defective cell proliferation, and early embryonic

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lethality in mice lacking tsg101. Proc. Natl. Acad. Sci. U. S. A. 98, 1859–1864 111 Miura, S. et al. (2000) Hgs (Hrs), a FYVE domain protein, is involved in Smad signaling through cooperation with SARA. Mol. Cell. Biol. 20, 9346–9355 112 Huang, X. et al. (2000) Structure of a WW domain containing fragment of dystrophin in complex with beta-dystroglycan. Nat. Struct. Biol. 7, 634–638

PTEN and myotubularin phosphatases: from 3-phosphoinositide dephosphorylation to disease Matthew J. Wishart and Jack E. Dixon The phosphatase and tensin homolog deleted on chromosome ten (PTEN) and myotubularin (MTM1) represent subfamilies of protein tyrosine phosphatases whose principal physiological substrates are D3-phosphorylated inositol phospholipids. As lipid phosphatases, PTEN- and MTM1-related (MTMR) proteins dephosphorylate the products of phosphoinositide 3-kinases and antagonize downstream effectors that utilize 3-phosphoinositides as ligands for protein targeting domains or allosteric activation. Here, we describe the cellular mechanisms of PTEN and MTMR function and their role in the etiology of cancer and other human diseases. Published online: 5 November 2002

Matthew J. Wishart Jack E. Dixon* Current address for both authors: 4433 Medical Sciences I, Box 0606, Dept of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109-0606, USA. Future address for both authors: Depts of Pharmacology and Cellular and Molecular Medicine, University of California, San Diego, Medical School, La Jolla, California 92093-0068, USA. *e-mail: jedixon@ umich.edu

Interconversion of phosphorylated inositol lipids is a fundamental eukaryotic signaling mechanism for eliciting an intracellular response to environmental changes. Phosphatidylinositol (PtdIns) is the most abundant membrane inositol lipid under resting conditions. In addition, it serves as the basic building block for differentially phosphorylated derivatives, which, together with PtdIns, are known as phosphoinositides (PI) [1]. In response to extracellular stimulation, specific PtdIns kinases phosphorylate positions 3-, 4- and 5- of the inositol head group to create seven unique phosphoinositides (Fig. 1). Many of these phosphoinositides function as ligands for protein targeting modules, such as pleckstrin homology (PH), phox homology (PX), epsin N-terminal homology (ENTH), band 4.1/ezrin/radixin/moesin (FERM) and Fab1p/YOTB/Vac1p/EEA1 (FYVE) domains (Fig. 1), where lipid binding leads to the translocation of their associated effector domains to membrane sites of protein complex formation, and, in certain cases, to allosteric regulation of target protein activity [2]. Subsequent dephosphorylation of the inositol head group by PtdIns-specific phosphatases removes these http://tcb.trends.com

targeting and allosteric regulatory signals. In this way, changes in PtdIns kinase and phosphatase activity in response to extracellular stimuli can dramatically alter the relative levels of specific PtdIns species and thereby orchestrate the spatio-temperal localization of proteins that regulate cellular response to its environment [1]. Conversely, dysregulation of PtdIns kinases and phosphatases results in aberrant phosphoinositide signaling and the disruption of normal control of cellular functions, such as growth, proliferation, differentiation and survival that are the hallmarks of disease. While much is known about the synthesis of PIs, their downstream effectors and associated signaling pathways [1], this review focuses on the regulation of PtdIns signaling by two families of 3-phosphoinositide phosphatases, PTEN and MTM, with particular emphasis on their role in the development of cancers and other human diseases. Function of 3-phosphoinositides

Synthesis of 3-phosphorylated inositol lipids is a crucial regulatory step in PtdIns metabolism (reviewed in [1]). PtdIns(3)P comprises 2–5% of singly phosphorylated cellular PtdIns, whereas very little of the other 3-PIs are present under resting conditions. Upon stimulation, three subclasses of PtdIns 3-kinases synthesize PtdIns(3)P, PtdIns(3,4)P2 and PtdIns(3,4,5)P3 by direct phosphorylation at the inositol D3 position of precursor lipids, while PtdIns(3,5)P2 is produced by PtdIns 5-kinase phosphorylation of PtdIns(3)P. Unlike other PIs, 3-phosphoinositides are not broken down to diacylglycerol and soluble inositol second messengers by PtdIns-specific phospholipase C. Instead, these lipids remain partitioned to membranes, where they recruit 3-PtdIns-specific binding domains to discrete subcellular locations.

0962-8924/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S0962-8924(02)02412-1

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Fig. 1. Enzymes and effectors governing 3-phosphoinositide metabolism and function. Arrows indicate major pathways for the synthesis and breakdown of 3-phosphoinositides. Classes of phosphoinositide 3-kinase (PI3K I, II and III) and isoforms of myotubularin-related proteins (myotubularin, MTM1; MTMR1, R1; etc.) are indicated. Representative interactions between 3-phosphoinositides and subfamilies of effector lipid binding domains (pleckstrin homology, PH; phox, PX; Fab1p/YOTB/Vac1p/EEA1, FYVE) are shown.

An example of the importance of PtdIns-mediated subcellular targeting is found in the protein serine/threonine kinase Akt and associated PtdIns-dependent kinases (PDKs) [3]. Box 1. Does PTEN function as both a 3-phosphoinositide and protein phosphatase in vivo? Based on sequence similarity to the superfamily of protein tyrosine phosphatases (PTPs), PTEN was initially postulated to dephosphorylate proteinaceous phosphomonesters before its identification as a phosphoinositide 3-phosphatase (see main text). Indeed, preliminary biochemical characterization showed that recombinant PTEN possessed a low level of phosphatase activity towards protein and peptide phospho-serine, -threonine and -tyrosine residues, in vitro, akin to the so-called dual-specificity subclass of PTPs [a]. Moreover, the marked preference in vitro for the highly acidic tyrosine-phosphorylated peptide poly(Glu4)Tyr [a] led to the idea of acidic inositol phospholipids as physiologically relevant PTEN substrates. Despite mounting genetic and biochemical evidence that points to phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] as the primary substrate for PTEN, particularly in regard to the etiology of disease (see main text), there are reports of PTEN acting as a PTP towards focal adhesion kinase and adaptor protein Shc, with downstream effects on cellular motility and directionality (reviewed in [b]). Unfortunately, interpretation of these studies generally suffers from overexpression of PTEN protein and low or inconsistent levels of phosphatase activity [b]. By contrast, recent genetic data from Drosophila melanogaster cast further weight to the primacy of the lipid phosphatase function of PTEN, where reversal of deleterious effects seen in PTEN-deficient flies can be achieved through mutation of the PtdIns(3,4,5)P3-specific PH domain in Akt [c]. Thus the lipid phosphatase activity of PTEN, and not its putative PTP activity, likely accounts for all PTEN functions in this organism. References a Myers, M.P. et al. (1997) P-TEN, the tumor suppressor from human chromosome 10q23, is a dual-specificity phosphatase. Proc. Natl. Acad. Sci. U. S. A. 94, 9052–9057 b Yamada, K.M. and Araki, M. (2001) Tumor suppressor PTEN: modulator of cell signaling, growth, migration and apoptosis. J. Cell Sci. 114, 2375–2382 c Stocker, H. et al. (2002) Living with lethal PIP3 levels: viability of flies lacking PTEN restored by a PH domain mutation in Akt/PKB. Science 295, 2088–2091

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In response to mitogen or cytokine stimulation, PtdIns 3-kinase activation leads to the production of PtdIns(3,4,5)P3 and subsequent recruitment of the PH domains of both Akt and PDKs to the same plasma membrane compartments. The resulting colocalization of kinases facilitates PDK phosphorylation of Akt, which in turn initiates Akt kinase activity toward its substrates involved in cellular growth and survival. Conversely, cell-permeable inhibitors of PtdIns 3-kinases, such as wortmannin and LY294002, block Akt/PDK translocation and Akt activation by antagonizing the production of PtdIns(3,4,5)P3. PtdIns(3,4,5)P3 and other 3-PIs have similarly been shown to modulate the downstream targets of PtdIns 3-kinase, including Bruton’s tyrosine kinase, GTPase-activating protein 1m and others [1]. Genetic manipulation of PtdIns 3-kinase activities in model organisms have implicated 3-PIs in cellular processes such as growth, apoptosis, DNA synthesis, changes in the cytoskeleton and vesicular trafficking. Given the central role of 3-PtdIns signaling in directing the subcellular localization of proteins, it is perhaps surprising that mutations in PtdIns 3-kinase genes have not been linked to human disease. By contrast, one of the clearest examples of how disrupting 3-PtdIns signaling leads to human pathology was first provided by the discovery of a novel tumor suppressor and PtdIns phosphatase, PTEN. PTEN as more than just a tumor suppressor

PTEN, also known as ‘mutated in multiple advanced cancers’, MMAC1, and TGFβ-regulated and epithelial cell-enriched phosphatase, TEP1, was first identified as a tumor-suppressor gene localized on chromosome 10q23 [4,5]. Somatic mutations in PTEN are found at high frequencies in endometrial carcinomas and high-grade gliomas [4–6]. Germline mutations in PTEN are associated with the related disorders Cowden disease, Lhermitte–Duclos and Bannayan–Zonana syndromes, where, in addition to increased rates of specific cancers, these diseases present a variety of hyperplastic, disorganized and nonmalignant growths [6]. The gene product of PTEN was initially postulated to function as a protein tyrosine phosphatase (PTP), PTPs being characterized by an invariant PTP active site motif, CxxxxxR (CX5R) [4,5]. Although capable of dephosphorylating proteinaceous substrates (Box 1), PTEN has been shown to utilize 3-phosphorylated phosphoinositides as physiological substrates, particularly PtdIns(3,4, 5)P3 [7], and several lines of evidence suggest that its lipid phosphatase activity is essential for PTEN tumor suppression [8]. As a 3-phosphatase, PTEN has emerged as a principal antagonist of PtdIns 3-kinase signaling, where it attenuates signaling through 3-phosphoinositidedependent effectors such as Akt and PDK1. Thus, the loss of PTEN in humans is thought to result in tumor development primarily through defects in Akt-mediated cell-cycle control and apoptosis [8].

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Numerous transgenic studies in mouse support a role for PTEN in Akt-mediated tumorigenesis and disease. Mouse embryos deficient in PTEN (PTEN−/−) exhibit a disruption in developmental patterning and regions of increased cellular proliferation that result in death before birth [9,10]. By contrast, mice heterozygous for a functional PTEN allele (PTEN+/−) are viable but develop spontaneous tumors. Cells from PTEN nullizygous mice and PTEN-deficient tumor cell lines each exhibit elevated levels of PtdIns(3,4,5)P3 that correlate with increased activation of Akt [8]. Expression of wild-type PTEN in these cells reduces PtdIns(3,4,5)P3 levels and inhibits Akt and other targets of PtdIns 3-kinase. Moreover, forced expression of activated Akt reverses PTEN-dependent inhibition. Functional deletion in a tissue- or developmental stage-specific manner has also revealed a role for PTEN in regulating cell size and migration in mouse brain (reviewed in [11]). Conditional deletion of PTEN in neuronal brain cells causes an increase in cell numbers, decreased cell death and enlarged soma size, resulting in macrocephaly. Selective PTEN deletion during early brain development reveals a role in regulating the potency and proliferation of stem cells, while disruption of PTEN solely in postmitotic neurons causes enlargement of the cerebellum and seizures as a result of the progressive enlargement of soma size [11], as well as improper migration of neuronal and glial cell populations [12]. Since many aspects of the neuronal characteristics of these mutant mice are also observed in Lhermitte–Duclos disease patients, it appears likely that a disruption of PTEN expression is sufficient to cause this disease in humans [11]. By contrast, conditional disruption of PTEN expression is not sufficient for neoplastic transformation of neuronal and glial cells [12]. In addition to its effects on neuronal cell number and size, PTEN has recently been shown to also regulate cardiomyocyte hypertrophy and G-protein-coupled receptor signaling in vivo, suggesting a role in the etiology of cardiovascular disease [42]. New roles for PTEN orthologs have also emerged from the invertebrates Drosophila melanogaster and Caenorhabditis elegans, where PTEN antagonizes insulin-stimulated PtdIns 3-kinase effects on cell number and size in flies [13,14], and metabolism and longevity in nematodes [15,16]. Reversal of hyperglycemia in diabetic mice via inhibition of PTEN mRNA expression [17] indicates that the PtdIns 3-phosphatase activity of PTEN is a physiologically relevant target for modulating mammalian insulin signaling in disease. Finally, recent reports of PTEN-mediated sensing of chemoattractant gradients and control of directional migration in Dictyostelium discoideum [18,19] point to the increasing complexity of PTEN function as more than simply a tumor-suppressor gene. PTEN structure and regulation

PTEN orthologs from yeast to human share the catalytic active site sequence C-K/R-A/M-G-K-G-R http://tcb.trends.com

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within their PtdIns phosphatase domains [8]. C-terminal to the catalytic domain, metazoan PTENs contain a Ca2+-independent C2 domain, and all mammalian proteins encode PEST sequences and a PDZ-binding motif at the extreme C-terminus (Fig. 2) [8]. The evolutionary conservation of these modular domains and motifs suggests that they play an important role in regulating PTEN function. Several determinants of substrate specificity tailored to PtdIns(3,4,5)P3 binding have been revealed from the crystal structure of human PTEN [20]. In particular, an insertion in the TI loop of the phosphatase domain widens and extends the PTEN catalytic pocket relative to other PTPs, to provide space for the bulky PtdIns(3,4,5)P3 headgroup. In addition, histidine 93, as well as two invariant lysines within the CX5R active site sequence (Lys125 and Lys128, above), coordinates the D1 and D5 phosphate groups of the inositol ring. The crystal structure also shows an extensive interface between the lipid phosphatase and C2 domains, suggesting that the latter might regulate PTEN catalytic activity. The C2 domain of PTEN binds to membrane phospholipids in a Ca2+-independent manner, and substitution of residues in the C2 domain that are crucial for lipid binding inhibits the ability of PTEN to function as a tumor suppressor. These substitutions also mistarget the mutant protein in vitro. As mutations affecting this interface are also frequently found in cancer [8], it appears that the C2 domain plays a role in both targeting and positioning of the lipid phosphatase in the plasma membrane. Downstream of the phosphatase domain, mammalian PTENs contain tandem PEST sequences [8]; however, little is known about their role in PTEN function. By contrast, mammalian PTENs also contain a C-terminal PDZ-binding motif (Fig. 2) that appears to associate with a variety of PDZ-domaincontaining proteins [8,21,22]. Phosphorylation of the C-terminal tail of PTEN suggests additional modes of regulating phosphatase activity, PDZ-binding or other protein–protein interactions [23–25]. In addition to direct regulation of PTEN protein function, several elements in the PTEN promoter appear to regulate the level of protein expression, including binding sites for the tumor suppressors p53 [26], early growth response-1 [27] and the peroxisome proliferator-activator receptor [28]. Induction of PTEN expression by these transcription factors results in an increase in cell survival in response to reduced Akt activity. Recently, PTEN inhibition of PtdIns 3-kinase signaling has been implicated in a positive-feedback loop for protecting p53 degradation [29], thereby creating a cooperative network between tumor-suppressor proteins. Finally, several PTEN paralogs have been identified in human [30,31] that share sequence similarity throughout the lipid phosphatase domain (Fig. 2). In contrast to PTEN, these proteins contain multiple predicted transmembrane spanning regions

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Fig. 2. PTEN and myotubularin-related proteins in human. Proteins were identified as previously described [31]. Protein names and chromosomal positions of genes are designated as in GenBank and Human Genome Project (http://genome.ucsc.edu/). Protein domains and motifs were identified using SMART (http://smart. embl-heidelberg.de/) and Pfam (http://www.sanger. ac.uk/Software/Pfam/). Abbreviations: coil, coiled-coil; C2, protein kinase C-like 2; Dead-Pase, non-catalytic STYX/ dead-phosphatase domain; DENN, differentially expressed in neoplastic versus normal cells; FYVE, Fab1p/YOTB/Vac1p/ EEA1; PBM, PDZ-binding motif; phosphatase, PtdIns 3-phosphatase catalytic domain; PH, pleckstrin homology domain; PH-G, pleckstrin homology-GRAM domain; TM, transmembrane spanning.

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(Fig. 2), exhibit a unique pattern of tissue expression and subcellular localization and have not been shown to regulate Akt activity. Further work is required to demonstrate whether subtle differences in their active site sequences alter the in vivo substrate specificity of these proteins relative to PTEN [30]. Myotubularin phosphatases

Myotubularin-related (MTMR) inositol lipid phosphatases comprise the largest known group of dual-specificity PTP-like enzymes conserved from yeast to human [31,32]. Of the 13 MTMR genes in human, eight are predicted to be enzymatically active, whereas the remaining genes encode proteins with a variety of substitutions to catalytically essential residues (Figs 2 and 3) [31]. All active myotubularin-related proteins contain the extended PTP catalytic motif CSDGWDRT [31] and exhibit activity towards phosphatidylinositol 3-phosphate [PtdIns(3)P] in vitro and in vivo [33–35]. PtdIns(3)P has been implicated in mediating vesicular trafficking http://tcb.trends.com

and is known to function as a targeting motif for various lipid-binding modules including FYVE, PH and PX domains [1]. Although the specific physiological role(s) of MTMRs as regulators of intracellular PtdIns(3)P have yet to be identified, linkage of mutations in genes encoding MTMRs with human neuromuscular diseases underscore their importance in developmental signaling pathways [31,32]. Altered expression of two myotubularin-related genes is associated with human disease. Myotubularin (MTM1), located on chromosome Xq28, is mutated in the severe muscle disorder X-linked myotubular myopathy, which manifests clinically as hypotonia and generalized muscle weakness in newborn males [36]. Heterozygous female carriers of a disrupted MTM1 allele also experience limb girdle and facial weakness (Fig. 3) [37]. Muscle cells in myotubular myopathy patients have large centrally located nuclei [36], characteristic of myogenic arrest in the latter stages of differentiation and maturation following myotube formation. Mutations in the MTMR2 gene,

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Fig. 3. Associated diseases and phylogenic relationship for myotubularin-related genes in human, fly, nematode and yeast. Genes were compiled from whole genomic sequences and transcripts from Homo sapiens (Hs), Drosophila melanogaster (Dm) Caenorhabditis elegans (Ce) and Saccharomyces cerevisiae (Sc) as previously described [31]. Evolutionary relationships were inferred from protein sequences using Phylip algorithms (http://evolution.genetics. washington.edu/phylip. html). Arrows designate diseases or functions associated with individual myotubularin-related gene products.

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located on chromosome 11q21, have also recently been linked to the neurodegenerative disorder Charcot–Marie–Tooth disease type 4B (CMT4B) [38]. CMT4B is a genetically recessive demyelinating neuropathy that results from abnormally folded myelin sheaths and Schwann cell proliferation in peripheral nerves. Of note is the distinct disease pathologies resulting from mutations in MTM1 and MTMR2 that suggest they are not functionally redundant, despite the fact that they share ~64% amino acid identity, utilize the same physiologic substrate and have overlapping patterns of tissue expression. Although their specific cellular roles are not known, differences in the level of expression in differentiating cells and dissimilar subcellular localization of MTM1 and MTMR2 proteins are thought to reflect the utilization of distinct pools of PtdIns(3)P [34]. MTMR structural features

The crystal structure of an MTMR protein has yet to be described; however, the myotubularin family encodes several modular features in addition to the phosphatase domain, which likely mediate protein–protein and lipid interactions. The N-terminal region of all myotubularin family members contains a predicted PH domain, in what had previously been described as a region of similarity between glucosyltransferases, Rab-like GTPase activators and myotubularins (Fig. 2) [31]. Although the functional significance of this domain http://tcb.trends.com

has not been demonstrated for MTMRs, PH domains are known to bind to phosphoinositides and play a role in membrane targeting. A coiled-coil motif is also predicted toward the C-terminus of most MTMRs (Fig. 2), which might play a role in forming interactions with other protein effectors. Individual myotubularin family members have additional PtdIns-binding domains (FYVE and PH) or protein interaction motifs (PDZ binding) that might serve to target MTMR phosphatase activity to specific subcellular regions containing PtdIns(3)P. Dead myotubularins: SBF1 and 3-PAP

The most extensively characterized catalytically dead MTMR protein is the cytoplasmic Set-binding factor 1 (Sbf1), also known as MTMR5 (Figs 2 and 3) [31]. In addition to its dead-phosphatase domain, Sbf1 contains a lipid-targeting PH domain and sequence similarity to a Rab3 GDP–GTP exchange factor (Fig. 2) [39]. The function of Sbf1 appears to depend on the non-catalytic state of its phosphatase-like domain as conversion of active-site residues that abrogate activity, Leu to Cys and Ile to Arg [31,39], confers hydrolytic activity to mutant Sbf1 towards phosphotyrosine and phosphoserine substrates and suppresses the effect of native Sbf1 on differentiation and growth [31,39]. While the cellular function of Sbf1 is unknown, an ortholog from D. melanogaster is found within the polycomb and histone acetyltransferase complexes

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Acknowledgements We acknowledge colleagues whose important work on PTEN and myotubularin-related proteins could not be cited owing to the focus and brevity of this review. We thank Gregory Taylor, Soo-A Kim, Knut Torgersen, Panayiotis Vacratsis, Michael Begley and Jeanne Stuckey for unpublished insights into PTEN/MTM function. The authors were supported by grants from the NIH and the Walther Cancer Institute.

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on nuclear chromatin [31], and male mice deficient for Sbf1 protein are infertile and azoospermatic (Fig. 3) [39]. In mouse, the primary defect caused by the absence of Sbf1 is first observed as basolateral vacuoles within the Sertoli cells of the seminiferous tubule [39]. Germ cell differentiation is also disrupted during the transition from pachytene spermatocytes to round spermatids, possibly as a byproduct of Sertoli cell dysfunction [39]. The dramatic reproductive consequences of Sbf1 disruption in mouse might thus provide insight into one cause of idiopathic infertility in humans. Perhaps a clue into the molecular function of Sbf1 can be gleaned from the dead-MTMR protein 3-PAP (Figs 2 and 3), which is postulated to bind to and activate D3-specific lipid phosphatase activity from cellular complexes [40]. Likewise, subcellular colocalization of MTM1 with the dead-MTMR Lip-STYX [34,41] suggests that MTMR-like dead-phosphatases might act in conjunction with active MTMRs to regulate levels of PtdIns 3-phosphates. Concluding remarks

The rapid pace of studies linking PTEN expression with human disease has placed this tumor suppressor alongside p53 as an essential cellular gatekeeper of

References 1 Vanhaesebroeck, B. et al. (2001) Synthesis and function of 3-phosphorylated inositol lipids. Annu. Rev. Biochem. 70, 535–602 2 Cullen, P.J. et al. (2001) Modular phosphoinositide-binding domains – their role in signalling and membrane trafficking. Curr. Biol. 11, R882–R893 3 Brazil, D.P. and Hemmings, B.A. (2001) Ten years of protein kinase B signaling: a hard Akt to follow. Trends Biochem. Sci. 26, 657–664 4 Li, J. et al. (1997) PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 275, 1943–1947 5 Steck, P.A. et al. (1997) Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers. Nat. Genet. 15, 356–362 6 Waite, K.A. and Eng, C. (2002) Protean PTEN: form and function. Am. J. Hum. Genet. 70, 829–844 7 Maehama, T. and Dixon, J.E. (1998) The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. J. Biol. Chem. 273, 13375–13378 8 Maehama, T. et al. (2001) PTEN and myotubularin: novel phosphoinositide phosphatases. Annu. Rev. Biochem. 70, 247–279 9 Di Cristofano, A. et al. (1998) Pten is essential for embryonic development and tumour suppression. Nat. Genet. 19, 348–355 10 Suzuki, A. et al. (1998) Male infertility, impaired spermatogenesis, and azoospermia in mice deficient for the pseudophosphatase Curr. Biol. 22, 1169–1178 11 Morrison, S.J. (2002) Pten-uating neural growth. Nat. Med. 8, 16–18 12 Marino, S. et al. (2002) PTEN is essential for cell migration but not for fate determination and tumourigenesis in the cerebellum. Development 129, 3513–3522 http://tcb.trends.com

proliferation, apoptosis and chemotaxis. Identification of PTEN as a PtdIns 3-phosphatase has renewed the emphasis in revealing downstream effectors of PtdIns 3-kinase signaling, their involvement in the development of disease and likelihood of being targets for drug therapy in the absence of PTEN expression. Future work should focus on the transcription, posttranscriptional processing, localization and protein interactions of PTEN to provide a better understanding of how its intrinsic PtdIns 3-phosphatase activity integrates into normal physiology as well as disease. Likewise, mutation of MTM1, MTMR2 and the catalytically dead MTMR and Sbf1 has been linked to pathologies of cellular maturation and differentiation. Although the precise role of these proteins has yet to be described, ongoing work on the mechanisms of MTMR regulation will likely reveal new aspects of regulating PtdIns-dependent cellular trafficking. Based on the conserved nature of MTMR structure and substrate specificity in organisms from yeast to human, future efforts are likely to be placed on linking mutations in other MTMR genes, including the catalytically dead forms, with idiopathic human diseases.

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Review

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Kinesin motors and disease Eckhard Mandelkow and Eva-Maria Mandelkow Kinesins are motor proteins that move cargoes such as vesicles, organelles and chromosomes along microtubules. They are best known for their role in axonal transport and in mitosis. There is a diverse family of kinesins, members of which differ in composition and functions. Roles of kinesins in diseases typically involve defective transport of cell components, transport of pathogens, or cell division. Published online: 5 November 2002

Eckhard Mandelkow* Eva-Maria Mandelkow Max-Planck-Unit for Structural Molecular Biology, Notkestrasse 85, 22607 Hamburg, Germany. *e-mail: [email protected]

Kinesin motor proteins generate motion along microtubules. During the past 17 years more than 1600 publications have appeared on kinesin that reveal an increasing variety of members in this superfamily of proteins with many possessing correspondingly diverse functions. Major functions include: • Movement of cargoes through the cytoplasm on microtubule tracks – cargoes include vesicles (e.g. Golgi-derived vesicles), organelles (mitochondria, peroxisomes, lysosomes), cytosolic components (e.g. mRNAs, proteins, elements of the cytoskeleton such as intermediate filaments, or virus particles) and membraneassociated complexes (e.g. rafts or intraflagellar transport particles). • Organization of the mitotic spindle – focusing of microtubules on spindle poles, attachment and movement of chromosomes, antiparallel sliding of microtubules during spindle elongation, regulation of microtubule dynamics. This review focuses on aspects of kinesin functions and interactions that are related to human diseases. Consistent with the motor and mitotic functions of kinesins, most disease-related aspects can be broadly classified as cases where physiological cargoes are not delivered appropriately (e.g. clogging of axonal transport); cases where non-physiological cargoes make use of the transport system (e.g. viruses); and cases where kinesins participate in mitosis and are therefore drug targets in cancer chemotherapy. http://tcb.trends.com

Kinesin structure, composition and nomenclature

The defining criterion for a kinesin is its ‘motor domain’, ≈320 residues in size, which binds and hydrolyses ATP (the energy source for movement) and binds to microtubules (the ‘tracks’ for movement; Fig. 1). The folding of the motor domain produces a core similar to that of other motors (e.g. the actin-dependent motor myosin) or signalling G-proteins [1]. The motor domain is usually coupled to additional domains with structural or regulatory roles. These in turn can attach to other cofactors, adaptor proteins or interaction modules [2]. The founding member, ‘conventional’ kinesin (alias KIF5/kinesin-I), contains an N-terminal motor domain, an α-helical stalk and a C-terminal tail domain (Fig. 1). The stalk of this ‘kinesin heavy chain’ (KHC) dimerizes with another KHC through a coiled-coil interaction. The two motor domains can ‘walk’ along a microtubule in a hand-over-hand fashion, alternately hydrolysing ATP and ensuring that the motion takes place processively without interruption [3] (see Box 1 for URLs to movies). This is achieved by major rearrangements in the motor/neck configuration on the microtubule surface [4,5]. Each KHC tail binds to a ‘kinesin light chain’ (KLC) as a cofactor, thus forming a heterotetramer. The stalk can bend in a hairpin fashion so that the tail interacts with the motor and inactivates it. This is one method of avoiding futile consumption of ATP while the motor is not needed [6]. The light chains contain tetratricopeptide repeat protein-interaction motifs (TPRs), which can dock onto adaptors or cargo receptors, linking kinesin to the cargo to be transported. There are many variations of this theme: other kinesins can be monomeric (KIF1); heterotrimeric (KIF3/kinesin-II, containing two distinct heavy chains, KIF3A and KIF3B, and one light chain, KAP3); or homotetrameric (two antiparallel pairs of heavy chain dimers, KIF11/Eg5). The interaction partners and docking complexes are only partially known, but this is an area of rapid

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