The nonconserved N-terminus of protein phosphatase 2B confers its properties to protein phosphatase 1

The protein phosphatase 1 catalytic subunit (PP1c) and the protein phosphatase 2B (PP2B or calcineurin) catalytic subunit (CNA) contain nonconserved N-terminal regions followed by conserved phosphatase cores. To examine the role of the N-termini of these two phosphatases, we substituted the residues 1-8 of PP1c with residues 1-42 of CNA, which is designated CNA(1-42)-PP1(9-330). The activities of CNA(1-42)-PP1(9-330) were similar to those of PP2B and different from those of PP1. The chimera was at least fourfold less sensitive to inhibition by okadaic acid, but was stimulated by nickel ions and chlorogenic acid, characteristics of PP2B not of PP1. These observations suggest that the N-terminus of CNA shifts the properties of PP1 toward those of PP2B. Our findings provide evidence that the nonconserved N-terminus of PP2B not only functions as important regulatory domain but also confers itself particular characteristics. This region may be targeted for regulation of PP2B activities in vivo.     

Identification of amino acids in the tetratricopeptide repeat and C-terminal domains of protein phosphatase 5 involved in autoinhibition and lipid activation

Protein phosphatase 5 (PP5) exhibits low basal activity due to the autoinhibitory properties of its N-terminal and C-terminal domains but can be activated approximately 40-fold in vitro by polyunsaturated fatty acids. To identify residues involved in regulating PP5 activity, we performed scanning mutagenesis of its N-terminal tetratricopeptide repeat (TPR) domain and deletion mutagenesis of its C-terminal domain. Mutating residues in a groove of the TPR domain that binds to heat shock protein 90 had no effect on basal phosphatase activity. Mutation of Glu-76, however, whose side chain projects away from this groove, resulted in a 10-fold elevation of basal activity without affecting arachidonic acid-stimulated activity. Thus, the interface of the TPR domain involved in PP5 autoinhibition appears to be different from that involved in heat shock protein 90 binding. We also observed a 10-fold elevation of basal phosphatase activity upon removing the C-terminal 13 amino acids of PP5, with a concomitant 50% decrease in arachidonic acid-stimulated activity. These two effects were accounted for by two distinct amino acid deletions: deleting the four C-terminal residues (496-499) of PP5 had no effect on its activity, but removing Gln-495 elevated basal activity 10-fold. Removal of a further three amino acids had no additional effect, but deleting Asn-491 resulted in a 50% reduction in arachidonic acid-stimulated activity. Thus, Glu-76 in the TPR domain and Gln-495 at the C-terminus were implicated in maintaining the low basal activity of PP5. While the TPR domain alone has been thought to mediate fatty acid activation of PP5, our data suggest that Asn-491, near its C-terminus, may also be involved in this process.     

Mutational analysis of Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP)

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) is a member of the serine/threonine protein phosphatases and shares 29% sequence identity with protein phosphatase 2Calpha (PP2Calpha) in its catalytic domain. To investigate the functional domains of CaMKP, mutational analysis was carried out using various recombinant CaMKPs expressed in Escherichia coli. Analysis of N-terminal deletion mutants showed that the N-terminal region of CaMKP played important roles in the formation of the catalytically active structure of the enzyme, and a critical role in polycation stimulation. A chimera mutant, a fusion of the N-terminal domain of CaMKP and the catalytic domain of PP2Calpha, exhibited similar substrate specificity to CaMKP but not to PP2Calpha, suggesting that the N-terminal region of CaMKP is crucial for its unique substrate specificity. Point mutations at Arg-162, Asp-194, His-196, and Asp-400, highly conserved amino acid residues in the catalytic domain of PP2C family, resulted in a significant loss of phosphatase activity, indicating that these amino acid residues may play important roles in the catalytic activity of CaMKP. Although CaMKP(1-412), a C-terminal truncation mutant, retained phosphatase activity, it was found to be much less stable upon incubation at 37 degrees C than wild type CaMKP, indicating that the C-terminal region of CaMKP is important for the maintenance of the catalytically active conformation. The results suggested that the N- and C-terminal sequences of CaMKP are essential for the regulation and stability of CaMKP.     

Protein phosphatase type 1 directs chitin synthesis at the bud neck in Saccharomyces cerevisiae

Yeast chitin synthase III (CSIII) is targeted to the bud neck, where it is thought to be tethered by the septin-associated protein Bni4. Bni4 also associates with the yeast protein phosphatase (PP1) catalytic subunit, Glc7. To identify regions of Bni4 necessary for its targeting functions, we created a collection of 23 deletion mutants throughout the length of Bni4. Among the deletion variants that retain the ability to associate with the bud neck, only those deficient in Glc7 binding fail to target CSIII to the neck. A chimeric protein composed of the septin Cdc10 and the C-terminal Glc7-binding domain of Bni4 complements the defects of a bni4Delta mutant, indicating that the C-terminus of Bni4 is necessary and sufficient to target Glc7 and CSIII to the bud neck. A Cdc10-Glc7 chimera fails to target CSIII to the bud neck but is functional in the presence of the C-terminal Glc7-binding domain of Bni4. The conserved C-terminal PP1-binding domain of mammalian Phactr-1 can functionally substitute for the C-terminus of Bni4. These results suggest that the essential role of Bni4 is to target Glc7 to the neck and activate it toward substrates necessary for CSIII recruitment and synthesis of chitin at the bud neck.     

The role of the C-terminal domain of protein tyrosine phosphatase-1B in phosphatase activity and substrate binding

Protein tyrosine phosphatase 1B (PTP-1B) has been implicated in the regulation of the insulin receptor. Dephosphorylation of the insulin receptor results in decreased insulin signaling and thus decreased glucose uptake. PTP-1B-/- mice have increased insulin sensitivity and are resistant to weight gain when fed a high fat diet, validating PTP-1B as a potential target for the treatment of type 2 diabetes. Many groups throughout the world have been searching for selective inhibitors for PTP-1B, and most of them target inhibitors to PTP-1B-(1-298), the N-terminal catalytic domain of the enzyme. However, the C-terminal domain is quite large and could influence the activity of the enzyme. Using two constructs of PTP-1B and a phosphopeptide as substrate, steady state assays showed that the presence of the C-terminal domain decreased both the Km and the k(cat) 2-fold. Pre-steady state kinetic experiments showed that the presence of the C-terminal domain improved the affinity of the enzyme for a phosphopeptide 2-fold, primarily because the off-rate was slower. This suggests that the C-terminal domain of PTP-1B may contact the phosphopeptide in some manner, allowing it to remain at the active site longer. This could be useful when screening libraries of compounds for inhibitors of PTP-1B. A compound that is able to make contacts with the C-terminal domain of PTP-1B would not only have a modest improvement in affinity but may also provide for specificity over other phosphatases.     

Crystal structure of the protein serine/threonine phosphatase 2C at 2.0 A resolution.

Protein phosphatase 2C (PP2C) is a Mn2+- or Mg2+-dependent protein Ser/Thr phosphatase that is essential for regulating cellular stress responses in eukaryotes. The crystal structure of human PP2C reveals a novel protein fold with a catalytic domain composed of a central beta-sandwich that binds two manganese ions, which is surrounded by alpha-helices. Mn2+-bound water molecules at the binuclear metal centre coordinate the phosphate group of the substrate and provide a nucleophile and general acid in the dephosphorylation reaction. Our model presents a framework for understanding not only the classical Mn2+/Mg2+-dependent protein phosphatases but also the sequence-related domains of mitochondrial pyruvate dehydrogenase phosphatase, the Bacillus subtilus phosphatase SpoIIE and a 300-residue domain within yeast adenyl cyclase. The protein architecture and deduced catalytic mechanism are strikingly similar to the PP1, PP2A, PP2B family of protein Ser/Thr phosphatases, with which PP2C shares no sequence similarity, suggestive of convergent evolution of protein Ser/Thr phosphatases.     

The metabotropic glutamate receptor mGluR7b binds to the catalytic gamma-subunit of protein phosphatase 1

Correct targeting of enzymes represents an important biological mechanism to control post-translational modifications of neurotransmitter receptors. The metabotropic glutamate receptor type 7 (mGluR7) exists in two splice variants (mGluR7a and mGluR7b), defined by different C-termini that are phosphorylated by protein kinase C (PKC). Recently, the search for mGluR7a binding partners yielded several proteins that interacted with its C-terminus. Here, a yeast two-hybrid screen using the mGluR7b C-terminus identified both variants of the catalytic gamma-subunit of protein phosphatase 1 (PP1gamma1 and PP1gamma2) as binding partners. The minimal interacting region of PP1gamma1/2 contained the core domain and was homologous to a region of PP1alpha that is needed for functional expression. Although this core domain is highly conserved within the protein phosphatase family, PP1alpha1 and PP1beta did not interact with mGluR7b. Binding between PP1gamma1 and mGluR7b might be regulated by alternative splicing, as the variant-specific distal part of the mGluR7b C-terminus mediated the interaction. Within this domain, amino acids involved in the binding to PP1gamma1 were mapped and biochemical assays using recombinant and native proteins verified the proposed interaction. Finally, the expression pattern of PP1gamma1, PP1gamma2 and mGluR7b was analysed in various CNS regions. In summary, these results suggest a regulation of mGluR7b by PP1gamma.     

Identification and characterization of the core region of protein phosphatase-1

PP1, PP2A and PP2B all belong to PPP family of serine/threonine protein phosphatases. Their primary structures are highly conserved, particularly in the catalytic domain. In order to obtain correlative information about this conserved region, we constructed N-, C-deletion and N/C double-deletion mutants. We found that the N- and Csingle-deletion mutants exhibited higher enzymatic activities, while specific activity of N/C double-deletion mutant PP1 (9-306) did not notably change. The results of kinetics analysis showed that k_cat and k_cat/K_m increased about 16-fold in the single-deletion mutants; while the two parameters of the double-deletion were lower than the single-deletions. We further explored stability of all mutants in existing denaturant guanidine hydrochloride (GdnHCl). It was noticeable that stability of PP1-(9-306) in all mutants was the highest. We speculated that PP1-(9-306) maybe retains a compact spherical structure, thus accordingly affected molecular catalysis. On the other hand the structures of single-deletion mutants were relatively relaxed, which were able to bind substrate easily, so activities of single-deletion mutants were higher than that of double-deletion mutant. We therefore deduced that PP1-(9-306) may be close to core region of PP1 molecule. In order to further solidify this idea, we used fluorescence spectra method to explore changes of space conformation. We found that emission peaks of all single-deletions were blue shifted in different degree in the absence of denaturant, while emission peak of N/C double-deletion mutant did not change obviously compared with that of the wild-type PP1. Conformation change of N/C double-deletion mutant was significantly less than those of single-deletion mutants in different GdnHCl concentration.     

Binding of phosphatase-1 delta to the retinoblastoma protein pRb involves domains that include substrate recognition residues and a pRB binding motif

Protein Ser/Thr phosphatase-1 (PP1) controls the retinoblastoma protein (pRb) function, including its dephosphorylation at mitotic exit. Since PP1delta was found to coimmunoprecipitate with pRb from mitotic and early G1 cells, we further investigated the PP1delta-pRb association using GST-full length and GST-deletion mutants of delta. GST-delta pulled-down pRb from G2, mitotic and G1 HeLa cells, thus confirming the coimmunoprecipitation results. Among the delta deletion mutants tested, pRb was pulled down by mutant 159-295, which reproduces the C-terminal domain of delta without the C-terminus, whereas the C-terminus alone did not pull-down pRb. Further fragmentation of the 159-295 mutant indicated that pRb was pulled down by fragment 195-260, which includes several residues involved in substrate binding, and by fragment 159-212, which contains the putative pRb-binding motif LxSxE. Altogether the results supported the hypothesis that PP1delta may contribute to the dephosphorylation of pRb at mitotic exit and that the PP1delta-pRb interaction may be at multiple sites.     

Protein phosphatase 2A scaffolding subunit A interacts with plasma membrane H+-ATPase C-terminus in the same region as 14-3-3 protein

The activity of the plant plasma membrane H⁺-ATPase is tightly regulated via phosphorylation and binding of 14-3-3 protein to the C-terminus of the pump. Whereas the 14-3-3-binding mechanism has been described in detail, the identity of specific protein kinases and phosphatases involved in the control of 14-3-3 binding has remained elusive. Using the yeast two-hybrid system, GST pull-down assays and overlay experiments, we report that scaffolding subunit A of protein phosphatase 2A (PP2A-A) interacts with the C-terminus of the Arabidopsis plasma membrane H⁺-ATPase isoform 2. PP2A-A binding is inhibited in the presence of 14-3-3 protein and fusicoccin, a fungal toxin which induces binding of 14-3-3 protein to the C-terminal end of the plasma membrane H⁺-ATPase. This indicates that PP2A-A and 14-3-3 protein compete with each other for binding to the same region in the C-terminus of the H⁺-ATPase.     

Importance of a surface hydrophobic pocket on protein phosphatase-1 catalytic subunit in recognizing cellular regulators

Cellular functions of protein phosphatase-1 (PP1), a major eukaryotic serine/threonine phosphatase, are defined by the association of PP1 catalytic subunits with endogenous protein inhibitors and regulatory subunits. Many PP1 regulators share a consensus RVXF motif, which docks within a hydrophobic pocket on the surface of the PP1 catalytic subunit. Although these regulatory proteins also possess additional PP1-binding sites, mutations of the RVXF sequence established a key role of this PP1-binding sequence in the function of PP1 regulators. WT PP1alpha, the C-terminal truncated PP1alpha-(1-306), a chimeric PP1alpha containing C-terminal sequences from PP2A, another phosphatase, PP1alpha-(1-306) with the RVXF-binding pocket substitutions L289R, M290K, and C291R, and PP2A were analyzed for their regulation by several mammalian proteins. These studies established that modifications of the RVXF-binding pocket had modest effects on the catalytic activity of PP1, as judged by recognition of substrates and sensitivity to toxins. However, the selected modifications impaired the sensitivity of PP1 to the inhibitor proteins, inhibitor-1 and inhibitor-2. In addition, they impaired the ability of PP1 to bind neurabin-I, the neuronal regulatory subunit, and G(M), the skeletal muscle glycogen-targeting subunit. These data suggested that differences in RVXF interactions with the hydrophobic pocket dictate the affinity of PP1 for cellular regulators. Substitution of a distinct RVXF sequence in inhibitor-1 that enhanced its binding and potency as a PP1 inhibitor emphasized the importance of the RVXF sequence in defining the function of this and other PP1 regulators. Our studies suggest that the diversity of RVXF sequences provides for dynamic physiological regulation of PP1 functions in eukaryotic cells.     

Structure of the bacteriophage lambda Ser/Thr protein phosphatase with sulfate ion bound in two coordination modes

The protein phosphatase encoded by bacteriophage lambda (lambda PP) belongs to a family of Ser/Thr phosphatases (Ser/Thr PPases) that includes the eukaryotic protein phosphatases 1 (PP1), 2A (PP2A), and 2B (calcineurin). These Ser/Thr PPases and the related purple acid phosphatases (PAPs) contain a conserved phosphoesterase sequence motif that binds a dinuclear metal center. The mechanisms of phosphoester hydrolysis by these enzymes are beginning to be unraveled. To utilize lambda PP more effectively as a model for probing the catalytic mechanism of the Ser/Thr PPases, we have determined its crystal structure to 2.15 A resolution. The overall fold resembles that of PP1 and calcineurin, including a conserved beta alpha beta alpha beta structure that comprises the phosphoesterase motif. Substrates and inhibitors probably bind in a narrow surface groove that houses the active site dinuclear Mn(II) center. The arrangement of metal ligands is similar to that in PP1, calcineurin, and PAP, and a bound sulfate ion is present in two novel coordination modes. In two of the three molecules in the crystallographic asymmetric unit, sulfate is coordinated to Mn2 in a monodentate, terminal fashion, and the two Mn(II) ions are bridged by a solvent molecule. Two additional solvent molecules are coordinated to Mn1. In the third molecule, the sulfate ion is triply coordinated to the metal center with one oxygen coordinated to both Mn(II) ions, one oxygen coordinated to Mn1, and one oxygen coordinated to Mn2. The sulfate in this coordination mode displaces the bridging ligand and one of the terminal solvent ligands. In both sulfate coordination modes, the sulfate ion is stabilized by hydrogen bonding interactions with conserved arginine residues, Arg 53 and Arg 162. The two different active site structures provide models for intermediates in phosphoester hydrolysis and suggest specific mechanistic roles for conserved residues.     

Growth arrest and DNA damage-inducible protein GADD34 assembles a novel signaling complex containing protein phosphatase 1 and inhibitor 1

The growth arrest and DNA damage-inducible protein, GADD34, was identified by its interaction with human inhibitor 1 (I-1), a protein kinase A (PKA)-activated inhibitor of type 1 protein serine/threonine phosphatase (PP1), in a yeast two-hybrid screen of a human brain cDNA library. Recombinant GADD34 (amino acids 233 to 674) bound both PKA-phosphorylated and unphosphorylated I-1(1-171). Serial truncations mapped the C terminus of I-1 (amino acids 142 to 171) as essential for GADD34 binding. In contrast, PKA phosphorylation was required for PP1 binding and inhibition by the N-terminal I-1(1-80) fragment. Pulldowns of GADD34 proteins expressed in HEK293T cells showed that I-1 bound the central domain of GADD34 (amino acids 180 to 483). By comparison, affinity isolation of cellular GADD34/PP1 complexes showed that PP1 bound near the C terminus of GADD34 (amino acids 483 to 619), a region that shows sequence homology with the virulence factors ICP34.5 of herpes simplex virus and NL-S of avian sarcoma virus. While GADD34 inhibited PP1-catalyzed dephosphorylation of phosphorylase a, the GADD34-bound PP1 was an active eIF-2alpha phosphatase. In brain extracts from active ground squirrels, GADD34 bound both I-1 and PP1 and eIF-2alpha was largely dephosphorylated. In contrast, the I-1/GADD34 and PP1/GADD34 interactions were disrupted in brain from hibernating animals, in which eIF-2alpha was highly phosphorylated at serine-51 and protein synthesis was inhibited. These studies suggested that modification of the I-1/GADD34/PP1 signaling complex regulates the initiation of protein translation in mammalian tissues.     

Apolipoprotein E and peptide mimetics modulate inflammation by binding the SET protein and activating protein phosphatase 2A

The molecular mechanism by which apolipoprotein E (apoE) suppresses inflammatory cytokine and NO production is unknown. Using an affinity purification approach, we found that peptide mimetics of apoE, derived from its receptor binding domain residues 130-150, bound to the SET protein, which is a potent physiological inhibitor of protein phosphatase 2A (PP2A). Both holo-apoE protein and apoE-mimetic peptides bound to the C-terminal region of SET, which is then associated with an increase in PP2A-mediated phosphatase activity. As physiological substrates for PP2A, the LPS-induced phosphorylation status of signaling MAPK and Akt kinase is reduced following treatment with apoE-mimetic peptides. On the basis of our previous report, in which apoE-mimetic peptides reduced I-κB kinase and NF-κB activation, we also demonstrate a mechanism for reduced production of inducible NO synthase protein and its NO product. These data provide evidence for a novel molecular mechanism by which apoE and apoE-mimetic peptides antagonize SET, thereby enhancing endogenous PP2A phosphatase activity, which reduces levels of phosphorylated kinases, signaling, and inflammatory response.     

Role of the 78-kDa glucose-regulated protein as an activity modulator of protein phosphatase1gamma2.

We have previously found the 78-kDa glucose-regulated protein (Grp78) to be a subunit of protein phosphatase1(PP1)gamma2. To determine the role of Grp78 in PP1gamma2 holoenzyme, we compared the two forms of this enzyme, PP1gamma2 holoenzyme containing Grp78 and Grp78-dissociated PP1gamma2 in rat testes in terms of their kinetic constants and sensitivities to inhibitors of this enzyme. The enzymatic activity of the Grp78-dissociated enzyme was much lower at whole range of concentrations of a substrate (phosphorylase a) than that of the holoenzyme; the Km value was about ten-fold higher in Grp78-dissociated enzyme than in holoenzyme, while the Vmax was similar. IC50s of the Grp78-dissociated enzyme for three inhibitors (microcystin-LR, inhibitor-2, and okadaic acid) were more than ten-fold higher than those of the holoenzyme. These results indicate that the Grp78 subunit modulates the activity of PP1gamma2 through its actions to control the binding of substrates or inhibitors to PP1gamma2.     

Interaction between protein phosphatase 5 and the A subunit of protein phosphatase 2A: evidence for a heterotrimeric form of protein phosphatase 5

Members of the phosphoprotein phosphatase family of serine/threonine phosphatases are thought to exist in different native oligomeric complexes. Protein phosphatase 2A (PP2A) is composed of a catalytic subunit (PP2Ac) that complexes with an A subunit, which in turn also interacts with one of many B subunits that regulate substrate specificity and/or (sub)cellular localization of the enzyme. Another family member, protein phosphatase 5 (PP5), contains a tetratricopeptide repeat domain at its N terminus, which has been suggested to mediate interactions with other proteins. PP5 was not thought to interact with partners homologous to the A or B subunits that exist within PP2A. However, our results indicate that this may not be the case. A yeast two-hybrid screen revealed an interaction between PP5 and the A subunit of PP2A. This interaction was confirmed for endogenous proteins in vivo using immunoprecipitation analysis and for recombinant proteins by in vitro binding experiments. Our results also indicate that the tetratricopeptide repeat domain of PP5 is required and sufficient for this interaction. In addition, immunoprecipitated PP5 contains associated B subunits. Thus, our results suggest that PP5 can exist in a PP2A-like heterotrimeric form containing both A and B subunits.     

Selective activators of protein phosphatase 5 target the auto-inhibitory mechanism

Protein phosphatase 5 (PP5) is an evolutionary conserved serine/threonine phosphatase. Its dephosphorylation activity modulates a diverse set of cellular factors including protein kinases and the microtubule-associated tau protein involved in neurodegenerative disorders. It is auto-regulated by its heat-shock protein (Hsp90)-interacting tetratricopeptide repeat (TPR) domain and its C-terminal α-helix. In the present study, we report the identification of five specific PP5 activators [PP5 small-molecule activators (P5SAs)] that enhance the phosphatase activity up to 8-fold. The compounds are allosteric modulators accelerating efficiently the turnover rate of PP5, but do barely affect substrate binding or the interaction between PP5 and the chaperone Hsp90. Enzymatic studies imply that the compounds bind to the phosphatase domain of PP5. For the most promising compound crystallographic comparisons of the apo PP5 and the PP5–P5SA-2 complex indicate a relaxation of the auto-inhibited state of PP5. Residual electron density and mutation analyses in PP5 suggest activator binding to a pocket in the phosphatase/TPR domain interface, which may exert regulatory functions. These compounds thus may expose regulatory mechanisms in the PP5 enzyme and serve to develop optimized activators based on these scaffolds.     

Function analysis of conserved amino acid residues in a Mn2+-dependent protein phosphatase, Pph3, from Myxococcus xanthus

The Myxococcus xanthus protein phosphatase Pph3 belongs to the Mg(2+)- or Mn(2+)-dependent protein phosphatase (PPM) family. Bacterial PPMs contain three divalent metal ions and a flap subdomain. Putative metal- or phosphate-ion binding site-specific mutations drastically reduced enzymatic activity. Pph3 contains a cyclic nucleotide monophosphate (cNMP)-binding domain in the C-terminal region, and it requires 2-mercaptoethanol for phosphatase activity; however, the C-terminal deletion mutant showed high activity in the absence of 2-mercaptoethanol. The phosphatase activity of the wild-type enzyme was higher in the presence of cAMP than in the absence of cAMP, whereas a triple mutant of the cNMP-binding domain showed slightly lower activities than those of wild-type, without addition of cAMP. In addition, mutational disruption of a disulphide bond in the wild-type enzyme increased the phosphatase activity in the absence of 2-mercaptoethanol, but not in the C-terminal deletion mutant. These results suggested that the presence of the C-terminal region may lead to the formation of the disulphide bond in the catalytic domain, and that disulphide bond cleavage of Pph3 by 2-mercaptoethanol may occur more easily with cAMP bound than with no cAMP bound.     

Molecular cloning in Arabidopsis thaliana of a new protein phosphatase 2C (PP2C) with homology to ABI1 and ABI2

We report the cloning of both the cDNA and the corresponding genomic sequence of a new PP2C from Arabidopsis thaliana, named AtP2C-HA (for homology to ABI1/ABI2). The AtP2C-HA cDNA contains an open reading frame of 1536 bp and encodes a putative protein of 511 amino acids with a predicted molecular mass of 55.7 kDa. The AtP2C-HA protein is composed of two domains, a C-terminal PP2C catalytic domain and a N-terminal extension of ca. 180 amino acid residues. The deduced amino acid sequence is 55% and 54% identical to ABI1 and ABI2, respectively. Comparison of the genomic structure of the ABI1, ABI2 and AtP2C-HA genes suggests that they belong to a multigene family. The expression of the AtP2C-HA gene is up-regulated by abscisic acid (ABA) treatment.     

Nuclear localization of protein phosphatase 5 is dependent on the carboxy-terminal region

Endogenous and overexpressed protein phosphatase 5 (PP5) localizes to the nucleus and cytoplasm of HeLa cells, while the overexpressed TPR domain of PP5 is restricted to the cytoplasm. Deletion and mutational analysis of human PP5 demonstrates that the C-terminal amino acids 420-499 are essential for nuclear localization and PP5 activity is not required. Since the phosphatase domain terminates at 473, these studies suggest that the highly conserved section (476-491) with the eukaryotic consensus FXAVPHPXPhiXPMAYAN is required for nuclear localization of PP5. Bacterially expressed PP5 is inhibited by several tumor promoters but not by the anticancer drug fostriecin.