Lafora disease (progressive myoclonus epilepsy of Lafora type) is an autosomal recessive neurodegenerative disorder resulting from defects in the EPM2A gene. EPM2A encodes a 331-amino acid protein containing a carboxyl-terminal phosphatase catalytic domain. We demonstrate that the EPM2A gene product also contains an amino-terminal carbohydrate binding domain (CBD) and that the CBD is critical for association with glycogen both in vitro and in vivo. The CBD domain localizes the phosphatase to specific subcellular compartments that correspond to the expression pattern of glycogen processing enzyme, glycogen synthase. Mutations in the CBD result in mis-localization of the phosphatase and thereby suggest that the CBD targets laforin to intracellular glycogen particles where it is likely to function. Thus naturally occurring mutations within the CBD of laforin likely result in progressive myoclonus epilepsy due to mis-localization of phosphatase expression.

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In Saccharomyces cerevisiae the CDC14 gene is essential for cell cycle progression. Strains carrying the cdc14-1 ts allele enter the cell cycle and arrest at restrictive temperatures. We have identified two human cDNAs encoding proteins which share sequence identity to the yeast CDC14p. The cell cycle arrest in cdc14-1 ts can be specifically complemented by the human cDNAs suggesting that they are functionally equivalent to the yeast CDC14 gene. Another clone identified in the search for human CDC14-like proteins corresponded to the putative tumor suppressor gene PTEN/MMAC1 (phosphatase and tensin homologue deleted on chromosome 10 or mutated in multiple advanced cancers 1). Analysis of the PTEN/MMAC1 showed that it did not complement the cdc14-1 ts allele and that it is more closely related to the yeast open reading frame YNL128W. Human CDC14p and PTEN/MMAC1 were expressed as recombinant proteins, and both were shown to have kinetic properties characteristic of dual specific phosphatases. The human CDC14p was localized in the nucleus while PTEN/MMAC1 has been reported to be localized in the cytoplasm. Our results suggest that CDC14 and YNL128W/PTEN/MMAC1 represent two related, but distinct, families of human and yeast phosphatases.

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Dual-specificity protein-tyrosine phosphatases (dsPTPases) have been implicated in the inactivation of mitogen-activated protein kinases (MAPKs). We have identified a novel phosphoserine/threonine/tyrosine-binding protein (STYX) that is related in amino acid sequence to dsPTPases, except for the substitution of Gly for Cys in the conserved dsPTPase catalytic loop (HCXXGXXR(S/ T)). cDNA subcloning and Northern blot analysis in mouse shows poly(A ؉ ) hybridization bands of 4.6, 2.4, 1.5, and 1.2 kilobases, with highest abundance in skeletal muscle, testis, and heart. Polymerase chain reaction amplification of reverse-transcribed poly(A ؉ ) RNA revealed an alternatively spliced form of STYX containing a unique carboxyl terminus. Bacterially expressed STYX is incapable of hydrolyzing Tyr(P)-containing substrates; however, mutation of Gly 120 to Cys (G120C), which structurally mimics the active site of dsPTPases, confers phosphatase activity to this molecule. STYX-G120C mutant hydrolyzes p-nitrophenyl phosphate and dephosphorylates both Tyr(P) and Thr(P) residues of peptide sequences of MAPK homologues. The kinetic parameters of dephosphorylation are similar to human dsPTPase, Vaccinia H1-related, including inhibition by vanadate. We believe this is the first example of a naturally occurring "dominant negative" phosphotyrosine/ serine/threonine-binding protein which is structurally related to dsPTPases. Dual-specificity phosphatases (dsPTPase)1 hydrolyze phosphoserine/threonine/tyrosine-containing substrates in vitro and exhibit a substrate preference in vitro and in vivo for diphosphorylated (Thr(P)/Tyr(P)) mitogen-activated protein kinase (MAPK) homologues (reviewed in Ref. 1). Most dsPTPases are localized to the nucleus (2-4), and it has been suggested that they are responsible for the nuclear dephosphorylation and inactivation of MAPKs seen in vivo (5). All dsPTPases contain the sequence, HCXXGXXR(S/T), which has been shown to correspond to the active site of PTPases (6). The essential cysteine forms a thiophosphate intermediate during dsPTPasecatalyzed dephosphorylation (7,8). Several investigators have shown that substitution of this Cys, by Ser, in the dsPTPases, abolishes hydrolytic activity in vitro (7, 9 -12) and function in vivo (10,13,14). Interestingly, transient expression of the Cys to Ser mutant prolongs MAPK activation in vivo (12), suggesting that it may compete with native phosphatases for binding to phosphorylated MAPK. Moreover, the mutant dsPTPase co-immunoprecipitates with phosphorylated MAPK (10), indicating that the catalytic cysteine is not essential for substrate binding. This conclusion is also supported by PTPase crystallographic studies showing that Cys to Ser mutants bind sulfate (15) and tyrosine-phosphorylated peptide (16) in a manner identical with native enzymes. This paper describes the identification and isolation of a novel phosphoserine/threonine/tyrosine-binding protein (STYX) that is related in amino acid sequence to dsPTPases, but contains a naturally occurring Gly residue in...