The structure and function of catalytic domains within inositol polyphosphate 5-phosphatases

Phosphoinositide signaling pathways regulate many essential cellular functions including proliferation, differentiation and survival, cytoskeletal organization, and vesicular trafficking. The inositol polyphosphate 5-phosphatases regulate the cellular levels of several bioactive phosphoinositide species. This review describes the structure and function of the 5-phosphatase and Sac1 catalytic domains of these enzymes. The crystal structure of the 5-phosphatase domain has been solved and shares homology with members of the AP endonuclease family. The phosphoinositide polyphosphatase activity of the Sac1 domain, found in some inositol polyphosphate 5-phosphatases, is defined by a motif, CX5 R(T/S), also found in both protein and lipid phosphatases.     

The inositol polyphosphate 5-phosphatases and the apurinic/apyrimidinic base excision repair endonucleases share a common mechanism for catalysis

Inositol polyphosphate 5-phosphatases (5-phosphatase) hydrolyze the 5-position phosphate from the inositol ring of phosphatidylinositol-derived signaling molecules; however, the mechanism of catalysis is only partially characterized. These enzymes play critical roles in regulating cell growth, apoptosis, intracellular calcium oscillations, and post-synaptic vesicular trafficking. The UCLA fold recognition server (threader) predicted that the conserved 300-amino acid catalytic domain, common to all 5-phosphatases, adopts the fold of the apurinic/apyrimidinic (AP) base excision repair endonucleases. PSI-BLAST searches of GENPEPT, using the amino acid sequence of AP endonuclease exonuclease III, identified all members of the 5-phosphatase family with highly significant scores. A sequence alignment between exonuclease III and all known 5-phosphatases revealed six highly conserved motifs containing residues that corresponded to the catalytic residues in the AP endonucleases. Mutation of each of these residues to alanine in the mammalian 43-kDa, or yeast Inp52p 5-phosphatase, resulted in complete loss of enzyme activity. We predict the 5-phosphatase enzymes share a similar mechanism of catalysis to the AP endonucleases, consistent with other common functional similarities such as an absolute requirement for magnesium for activity. Based on this analysis, functional roles have been assigned to conserved residues in all 5-phosphatase enzymes.     

Inositol 5-phosphatases: insights from the Lowe syndrome protein OCRL

The precise regulation of phosphoinositide lipids in cellular membranes is crucial for cellular survival and function. Inositol 5-phosphatases have been implicated in a variety of disorders, including various cancers, obesity, type 2 diabetes, neurodegenerative diseases and rare genetic conditions. Despite the obvious impact on human health, relatively little structural and biochemical information is available for this family. Here, we review recent structural and mechanistic work on the 5-phosphatases with a focus on OCRL, whose loss of function results in oculocerebrorenal syndrome of Lowe and Dent 2 disease. Studies of OCRL emphasize how the actions of 5-phosphatases rely on both intrinsic and extrinsic membrane recognition properties for full catalytic function. Additionally, structural analysis of missense mutations in the catalytic domain of OCRL provides insight into the phenotypic heterogeneity observed in Lowe syndrome and Dent disease.     

Phosphatidylinositol 3,4,5-trisphosphate is a substrate for the 75 kDa inositol polyphosphate 5-phosphatase and a novel 5-phosphatase which forms a complex with the p85/p110 form of phosphoinositide 3-kinase.

Agonist-stimulated production of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3], is considered the primary output signal of activated phosphoinositide (PI) 3-kinase. The physiological targets of this novel phospholipid and the identity of enzymes involved in its metabolism have not yet been established. We report here the identification of two enzymes which hydrolyze the 5-position phosphate of PtdIns(3,4,5)P3, forming phosphatidylinositol (3,4)-bisphosphate. One of these enzymes is the 75 kDa inositol polyphosphate 5-phosphatase (75 kDa 5-phosphatase), which has previously been demonstrated to metabolize inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. We have identified a second PtdIns(3,4,5)P3 5-phosphatase in the cytosolic fraction of platelets, which forms a complex with the p85/p110 form of PI 3-kinase. This enzyme is immunologically and chromatographically distinct from the platelet 43 kDa and 75 kDa 5-phosphatases and is unique in that it removes the 5-position phosphate from PtdIns(3,4,5)P3, but does not metabolize PtdIns(4,5)P2, Ins(1,4,5)P3 or Ins(1,3,4,5)P4. These studies demonstrate the existence of multiple PtdIns(3,4,5)P3 5-phosphatases within the cell.     

Regulation of phosphoinositide signaling by the inositol polyphosphate 5-phosphatases

Phosphoinositide signaling molecules control cellular growth, proliferation and differentiation, intracellular vesicle trafficking, and cytoskeletal rearrangement. The inositol polyphosphate 5-phosphatase family remove the D-5 position phosphate from PtdIns(3,4,5)P3, PtdIns(4,5)P2 and PtdIns(3,5)P2 forming PtdIns(3,4)P2, PtdIns(4)P and PtdIns(3)P respectively. This enzyme family, comprising ten mammalian members, exhibit seemingly non-redundant functions including the regulation of synaptic vesicle recycling, hematopoietic cell function and insulin signaling. Here we highlight recently established insights into the functions of two well characterized 5-phosphatases OCRL and SHIP2, which have been the subject of extensive functional studies, and the characterization of recently identified members, SKIP and PIPP, in order to highlight the diverse and complex functions of this enzyme family.     

Molecular characterization of an Arabidopsis gene encoding a phospholipid-specific inositol polyphosphate 5-phosphatase

Phosphoinositides are important molecules that serve as second messengers and bind to a complex array of proteins modulating their subcellular location and activity. The enzymes that metabolize phosphoinositides can in some cases serve to terminate the signaling actions of phosphoinositides. The inositol polyphosphate 5-phosphatases (5PTases) comprise a large protein family that hydrolyzes 5-phosphates from a variety of inositol phosphate and phosphoinositide substrates. We previously reported the identification of 15 putative 5PTase genes in Arabidopsis and have shown that overexpression of the At5PTase1 gene can alter abscisic acid signaling. At5PTase1 and At5PTase2 have been shown to hydrolyze the 5-phosphate from inositol phosphate substrates. We have examined the substrate specificity of the At5PTase11 protein, which is one of the smallest predicted 5PTases found in any organism. We report here that the At5PTase11 gene encodes an active 5PTase enzyme that can only dephosphorylate phosphoinositide substrates containing a 5-phosphate. In addition to hydrolyzing known substrates of 5PTase enzymes, At5PTase11 also hydrolyzes the 5-phosphate from phosphatidylinositol (3,5) bisphosphate. We also show that the At5PTase11 gene is regulated by abscisic acid, jasmonic acid, and auxin, suggesting a role for phosphoinositide action in these signal transduction pathways.     

Inositol polyphosphate 5-phosphatases 1 and 2 are required for regulating seedling growth

Signals can be perceived and amplified at the cell membrane by receptors coupled to the production of a variety of second messengers, including myoinositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)]. The myoinositol polyphosphate 5-phosphatases (5PTases; EC 3.1.3.56) comprise a large protein family that hydrolyzes 5-phosphates from a variety of myoinositol phosphate (InsP) and phosphoinositide phosphate (PtdInsP) substrates. Arabidopsis thaliana has 15 genes encoding 5PTases. Biochemical analyses of a subgroup of 5PTase enzymes suggest that these enzymes have both overlapping and unique substrate preferences. Ectopic expression of these genes in transgenic plants can reduce Ins(1,4,5)P(3) levels and alter abscisic acid (ABA) signaling. To further explore the function of 5PTases in signaling, we have identified and characterized T-DNA insertional mutants for 5PTase1 and 5PTase2 and produced a double mutant. When grown in the dark, the seeds from these mutants germinate faster than wild-type seeds and the mutant seedlings have longer hypocotyls than wild-type seedlings. Seeds from these mutant lines also demonstrate an increase in sensitivity to ABA. These changes in early seedling growth are accompanied by mass increases in Ins(1,4,5)P(3), but not by changes in endogenous ABA content. By labeling the endogenous myoinositol pool in 5ptase1 and 5ptase2 mutants, we detected increases in Ins(1,4,5)P(3) and a decrease in PtdIns, PtdIns(4)P, and phosphatidylinositol (4,5) bisphosphate. Taken together, these data indicate that the At5PTase1 and At5PTase2 genes have nonredundant roles in hydrolyzing inositol second-messenger substrates and that regulation of Ins(1,4,5)P(3) levels is important during germination and early seedling development.     

Inositol polyphosphate 5-phosphatases; new players in the regulation of cilia and ciliopathies

Phosphoinositides regulate numerous cellular events via the recruitment and activation of multiple lipid-binding effector proteins. The precise temporal and spatial regulation of phosphoinositide signals by the co-ordinated activities of phosphoinositide kinases and phosphatases is essential for homeostasis and development. Mutations in two inositol polyphosphate 5-phosphatases, INPP5E and OCRL, cause the cerebrorenal syndromes of Joubert and Lowe's, respectively. INPP5E and OCRL exhibit overlapping phosphoinositide substrate specificity and subcellular localisation, including an association with the primary cilia. Here, we review recent studies that identify a new role for these enzymes in the regulation of primary cilia function. Joubert syndrome has been extensively linked to primary cilia defects, and Lowe's may represent a new class of 'ciliopathy associated' syndromes.     

Soluble and particulate inositol 1,4,5-trisphosphate 5-phosphatases show common antigenic determinants

Inositol 1,4,5-trisphosphate 5-phosphatase catalyses the dephosphorylation of the phosphate in the 5-position from inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate. One particulate and two soluble enzymes were previously described in bovine brain. In this study, we have obtained a precipitating antiserum against soluble type I inositol 1,4,5-trisphosphate 5-phosphatase. The particulate, but not the soluble type II enzyme, was immunoprecipitated by the serum. Inositol 1,4,5-triphosphate 5-phosphatase activity from crude extracts of rat brain, human platelets and rat liver were immmunoprecipitated by the same antibodies, suggesting the existence of common antigenic determinant among inositol 1,4,5-trisphosphate 5-phosphatases of diverse sources.     

A diverse family of inositol 5-phosphatases playing a role in growth and development in Dictyostelium discoideum

Inositol phosphate-containing molecules play an important role in a broad range of cellular processes. Inositol 5-phosphatases participate in the regulation of these signaling molecules. We have identified four inositol 5-phosphatases in Dictyostelium discoideum, Dd5P1-4, showing a high diversity in domain composition. Dd5P1 possesses only a inositol 5-phosphatase catalytic domain. An unique domain composition is present in Dd5P2 containing a RCC1-like domain. RCC1 has a seven-bladed propeller structure and interacts with G-proteins. Dd5P3 and Dd5P4 have a domain composition similar to human Synaptojanin with a SacI domain and OCRL with a RhoGAP domain, respectively. We have expressed the catalytic domains and show that these inositol 5-phosphatases have different substrate preferences. Single and double gene inactivation suggest a functional redundancy for Dd5P1, Dd5P2, and Dd5P3. Inactivation of the gene coding for Dd5P4 leads to defects in growth and development. These defects are restored by the expression of the complete protein but not by the 5-phosphatase catalytic domain.     

The yeast inositol polyphosphate 5-phosphatases inp52p and inp53p translocate to actin patches following hyperosmotic stress: mechanism for regulating phosphatidylinositol 4,5-bisphosphate at plasma membrane invaginations

The Saccharomyces cerevisiae inositol polyphosphate 5-phosphatases (Inp51p, Inp52p, and Inp53p) each contain an N-terminal Sac1 domain, followed by a 5-phosphatase domain and a C-terminal proline-rich domain. Disruption of any two of these 5-phosphatases results in abnormal vacuolar and plasma membrane morphology. We have cloned and characterized the Sac1-containing 5-phosphatases Inp52p and Inp53p. Purified recombinant Inp52p lacking the Sac1 domain hydrolyzed phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] and PtdIns(3, 5)P(2). Inp52p and Inp53p were expressed in yeast as N-terminal fusion proteins with green fluorescent protein (GFP). In resting cells recombinant GFP-tagged 5-phosphatases were expressed diffusely throughout the cell but were excluded from the nucleus. Following hyperosmotic stress the GFP-tagged 5-phosphatases rapidly and transiently associated with actin patches, independent of actin, in both the mother and daughter cells of budding yeast as demonstrated by colocalization with rhodamine phalloidin. Both the Sac1 domain and proline-rich domains were able to independently mediate translocation of Inp52p to actin patches, following hyperosmotic stress, while the Inp53p proline-rich domain alone was sufficient for stress-mediated localization. Overexpression of Inp52p or Inp53p, but not catalytically inactive Inp52p, which lacked PtdIns(4,5)P(2) 5-phosphatase activity, resulted in a dramatic reduction in the repolarization time of actin patches following hyperosmotic stress. We propose that the osmotic-stress-induced translocation of Inp52p and Inp53p results in the localized regulation of PtdIns(3,5)P(2) and PtdIns(4,5)P(2) at actin patches and associated plasma membrane invaginations. This may provide a mechanism for regulating actin polymerization and cell growth as an acute adaptive response to hyperosmotic stress.     

Phosphatidylinositol 4-Kinases and PI4P Metabolism in the Nervous System: Roles in Psychiatric and Neurological Diseases

The four mammalian phosphatidylinositol 4-kinases, together with the PI(4,5)P₂ depleting 5-phosphatases of the oculocerebrorenal syndrome of Lowe and synaptojanin families, modulate neuronal pools of PI4P lipid and regulate intracellular membrane trafficking in the endocytic and secretory pathways. Dysfunctions in these enzymes have been associated with a broad spectrum of disorders including schizophrenia, bipolar disorder, Lowe syndrome, age-related neurodegeneration, Alzheimer’s disease and Down syndrome. Recent work has shown that reduced expression of individual phosphatidylinositol 4-kinase isozymes is associated with impaired survival of specific neuronal populations within the CNS. Furthermore, alterations to the concentrations of different phosphoinositide lipid species in the brain and, in particular, the ratio of PI4P to PI(4,5)P₂ can have deleterious effects on clathrin-dependent membrane trafficking both in the Golgi–endosomal pathway and at the plasma membrane. In this article, we focus on the cell biology, biochemistry and neuronal functions of the phosphatidylinositol 4-kinases and their emerging roles in psychiatric and neurological pathologies.     

Comparative mechanistic and substrate specificity study of inositol polyphosphate 5-phosphatase Schizosaccharomyces pombe Synaptojanin and SHIP2

Inositol-5-phosphatases are important enzymes involved in the regulation of diverse cellular processes from synaptic vesicle recycling to insulin signaling. We describe a comparative study of two representative inositol-5-phosphatases, Schizosaccharomyces pombe synaptojanin (SPsynaptojanin) and human SH2 domain-containing inositol-5-phosphatase SHIP2. We show that in addition to Mg2+, transition metals such as Mn2+, Co2+, and Ni2+ are also effective activators of SPsynaptojanin. In contrast, Ca2+ and Cu2+ are inhibitory. We provide evidence that Mg2+ binds the same site occupied by Ca2+ observed in the crystal structure of SPsynaptojanin complexed with inositol 1,4-bisphosphate (Ins(1,4)P2). Ionizations important for substrate binding and catalysis are defined for the SPsynaptojanin-catalyzed Ins(1,4,5)P3 reaction. Kinetic analysis with four phosphatidylinositol lipids bearing a 5-phosphate and 54 water-soluble inositol phosphates reveals that SP-synaptojanin and SHIP2 possess much broader substrate specificity than previously appreciated. The rank order for SPsynaptojanin is Ins(2,4,5)P3 > phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) approximately Ins(4,5)P2 approximately Ins(1,4,5)P3 approximately Ins(4,5,6)P3 > PtdIns(3,5)P2 approximately PtdIns(3,4,5)P3 approximately Ins(1,2,4,5)P4 approximately Ins(1,3,4,5)P4 approximately Ins-(2,4,5,6)P4 approximately Ins(1,2,4,5,6)P5. The rank order for SHIP2 is Ins(1,2,3,4,5)P5 > Ins(1,3,4,5)P4 > PtdIns(3,4,5)P4 approximately PtdIns(3,5)P2 approximately Ins(1,4,5,6)P4 approximately Ins(2,4,5,6)P4. Because inositol phosphate isomers elicit different biological activities, the extended substrate specificity for SPsynaptojanin and SHIP2 suggest that these enzymes likely have multiple roles in cell signaling and may regulate distinct pathways. The unique substrate specificity profiles and the importance of 2-position phosphate in binding also have important implications for the design of potent and selective SPsynaptojanin and SHIP2 inhibitors for pharmacological investigation.     

The myotubularin family of lipid phosphatases

The myotubularins (MTMs) constitute a large family of phosphoinositide lipid 3-phosphatases with specificity for PtdIns3P and PtdIns (3,5)P2. Mutations in MTM proteins are associated with inherited conditions such as myotubular myopathy and Charcot-Marie-Tooth syndrome. The substrate lipids are known to be regulators of the endosomal pathway through recruitment of specific effector proteins. Hydrolysis of PtdIns (3,5)P2 provides a biosynthetic pathway to the production of PtdIns5P, which itself can allosterically activate MTMs. We review the properties of this intriguing family of proteins and discuss potential physiological functions that include regulation of the endocytic pathway.     

A phosphatidylinositol phosphate-specific myo-inositol polyphosphate 5-phosphatase required for seedling growth

The phosphatidylinositol phosphate signaling pathway is involved in many crucial cellular functions. The myo-inositol polyphosphate 5-phosphatases (5PTases) (E.C. 3.1.3.56) comprise a large protein family that hydrolyze 5-phosphates from a variety of phosphatidylinositol phosphate and inositol phosphate substrates. We previously reported that the At5PTase11 enzyme (At1g47510), which is one of the smallest predicted 5PTases found in any organism, encodes an active 5PTase whose activity is restricted to tris- and bis-, but not mono-phosphorylated phosphatidylinositol phosphate substrates containing a 5-phosphate. This is in contrast to other unrestricted Arabidopsis 5PTases, which also hydrolyze tris- and bis inositol phosphate molecules. To further explore the function of At5PTase11, we have characterized two T-DNA mutants in the At5PTase11 gene, and have complemented this mutant. Seed from 5ptase11 mutants germinate slower than wildtype seed and mutant seedlings have decreased hypocotyl growth as compared to wildtype seedlings when grown in the dark. This phenotype is the opposite of the increased hypocotyl growth phenotype previously described for other 5ptase mutants defective in inositol phosphate-specific 5PTase enzymes. By labeling the endogenous myo-inositol pool in 5ptase11 mutants, we correlated these hypocotyl growth changes with a small increase in the 5PTase11 substrate, phosphatidylinositol (4,5) bisphosphate, and decreases in the potential products of 5PTase11, phosphatidylinositol (3) phosphate and phosphatidylinositol (4) phosphate. Surprisingly, we also found that dark-grown 5ptase11 mutants contain increases in inositol (1,4,5) trisphosphate and an inositol bisphosphate that is not a substrate for recombinant 5PTase11. We present a model for regulation of hypocotyl growth by specific molecules found in this pathway.     

The yeast inositol polyphosphate 5-phosphatase Inp54p localizes to the endoplasmic reticulum via a C-terminal hydrophobic anchoring tail: regulation of secretion from the endoplasmic reticulum

The budding yeast Saccharomyces cerevisiae has four inositol polyphosphate 5-phosphatase (5-phosphatase) genes, INP51, INP52, INP53, and INP54, all of which hydrolyze phosphatidylinositol (4,5)-bisphosphate. INP54 encodes a protein of 44 kDa which consists of a 5-phosphatase domain and a C-terminal leucine-rich tail, but lacks the N-terminal SacI domain and proline-rich region found in the other three yeast 5-phosphatases. We report that Inp54p belongs to the family of tail-anchored proteins and is localized to the endoplasmic reticulum via a C-terminal hydrophobic tail. The hydrophobic tail comprises the last 13 amino acids of the protein and is sufficient to target green fluorescent protein to the endoplasmic reticulum. Protease protection assays demonstrated that the N terminus of Inp54p is oriented toward the cytoplasm of the cell, with the C terminus of the protein also exposed to the cytosol. Null mutation of INP54 resulted in a 2-fold increase in secretion of a reporter protein, compared with wild-type yeast or cells deleted for any of the SacI domain-containing 5-phosphatases. We propose that Inp54p plays a role in regulating secretion, possibly by modulating the levels of phosphatidylinositol (4,5)-bisphosphate on the cytoplasmic surface of the endoplasmic reticulum membrane.     

Ins(1,4,5)P3 metabolism and the family of IP3-3Kinases

The release of Ca2+ from intracellular stores is triggered by the second messenger inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3). The regulation of this process is critically important for cellular homeostasis. Ins(1,4,5)P3 is rapidly metabolised, either to inositol (1,4)-bisphosphate (Ins(1,4)P2) by inositol polyphosphate 5-phosphatases or to inositol (1,3,4,5)-tetrakisphosphate (Ins(1,3,4,5)P4) by one of a family of inositol (1,4,5)P3 3-kinases (IP3-3Ks). Three isoforms of IP3-3K have now been identified in mammals; they have a conserved C-terminal catalytic domain, but divergent N-termini. This review discusses the metabolism of Ins(1,4,5)P3, compares the IP3-3K isoforms and addresses potential mechanisms by which their activity might be regulated.     

Myotubularin phosphatases: policing 3-phosphoinositides

In eukaryotic cells, phosphatidylinositol is subject to differential phosphorylation, resulting in the production of seven distinct phosphatidylinositol phosphates, often referred to as phosphoinositides (PIs). PIs have numerous distinct roles in cellular regulation and membrane trafficking. Recently, myotubularin family PI 3-phosphatases have emerged as key regulators of phosphatidylinositol 3-phosphate and phosphatidylinositol 3,5-bisphosphate, two PIs that regulate traffic within the endosomal-lysosomal pathway. Mutations in several myotubularin genes lead to myotubular myopathy and Charcot-Marie-Tooth peripheral neuropathy. Strikingly, nearly half of the members of the human myotubularin family appear to be catalytically inactive. Several inactive myotubularins have essential functions in mammals. Recent work in mammalian cells and model organisms is shedding light on the roles of myotubularins in membrane traffic.     

The Sac1 phosphoinositide phosphatase regulates Golgi membrane morphology and mitotic spindle organization in mammals

Phosphoinositides (PIPs) are ubiquitous regulators of signal transduction events in eukaryotic cells. PIPs are degraded by various enzymes, including PIP phosphatases. The integral membrane Sac1 phosphatases represent a major class of such enzymes. The central role of lipid phosphatases in regulating PIP homeostasis notwithstanding, the biological functions of Sac1-phosphatases remain poorly characterized. Herein, we demonstrate that functional ablation of the single murine Sac1 results in preimplantation lethality in the mouse and that Sac1 insufficiencies result in disorganization of mammalian Golgi membranes and mitotic defects characterized by multiple mechanically active spindles. Complementation experiments demonstrate mutant mammalian Sac1 proteins individually defective in either phosphoinositide phosphatase activity, or in recycling of the enzyme from the Golgi system back to the endoplasmic reticulum, are nonfunctional proteins in vivo. The data indicate Sac1 executes an essential household function in mammals that involves organization of both Golgi membranes and mitotic spindles and that both enzymatic activity and endoplasmic reticulum localization are important Sac1 functional properties.     

Conserved sequence motifs among bacterial,eukaryotic, and archaeal phosphatases that define a new phosphohydrolase superfamily.

Members of a new molecular family of bacterial nonspecific acid phosphatases (NSAPs), indicated as class C, were found to share significant sequence similarities to bacterial class B NSAPs and to some plant acid phosphatases, representing the first example of a family of bacterial NSAPs that has a relatively close eukaryotic counterpart. Despite the lack of an overall similarity, conserved sequence motifs were also identified among the above enzyme families (class B and class C bacterial NSAPs, and related plant phosphatases) and several other families of phosphohydrolases, including bacterial phosphoglycolate phosphatases, histidinol-phosphatase domains of the bacterial bifunctional enzymes imidazole-glycerolphosphate dehydratases, and bacterial, eukaryotic, and archaeal phosphoserine phosphatases and threalose-6-phosphatases. These conserved motifs are clustered within two domains, separated by a variable spacer region, according to the pattern [FILMAVT]-D-[ILFRMVY]-D-[GSNDE]-[TV]-[ILVAM]-[AT S VILMC]-X-¿YFWHKR)-X-¿YFWHNQ¿-X( 102,191)-¿KRHNQ¿-G-D-¿FYWHILVMC¿-¿QNH¿-¿FWYGP¿-D -¿PSNQYW¿. The dephosphorylating activity common to all these proteins supports the definition of this phosphatase motif and the inclusion of these enzymes into a superfamily of phosphohydrolases that we propose to indicate as "DDDD" after the presence of the four invariant aspartate residues. Database searches retrieved various hypothetical proteins of unknown function containing this or similar motifs, for which a phosphohydrolase activity could be hypothesized.