Sec-dependent and Sec-independent translocation of haloacid dehalogenase Chd1 of Burkholderia cepacia MBA4 in Escherichia coli

2-Haloacid dehalogenases are hydrolytic enzymes that cleave the halogen-carbon bond(s) in haloalkanoic acids. We have previously isolated a cryptic haloacid dehalogenase gene from Burkholderia cepacia MBA4 and expressed it in Escherichia coli. This recombinant protein is unusual in having a long leader sequence, a property of periplasmic enzymes. In this paper, we report the functional role of this leader sequence. Western blot analyses showed that Chd1 is translocated to the periplasm. The results on the expression of Chd1 in the presence of sodium azide suggested the cleavage of the leader to be Sec-dependent. Chimeras of Chd1 and green fluorescent protein demonstrated that the leader sequence is fully functional in translocating the fusion protein to the periplasm. The expression of the chimeras in Sec mutants supported the Sec-dependent translocation. Surprisingly, recombinant Chd1 and a chimera with no leader sequence were also found in the periplasm.     

Periplasmic location of the pesticin immunity protein suggests inactivation of pesticin in the periplasm.

The pesticin activity and immunity genes on plasmid pPCP1 of Yersinia pestis were sequenced. They encoded proteins of 40 kDa (pesticin) and 16 kDa (immunity protein); the latter was found in the periplasm. The location of the immunity protein suggests that imported pesticin is inactivated in the periplasm before it hydrolyzes murein. Pesticin contains a TonB box close to the N-terminal end that is identical to the TonB box of colicin B. The DNA sequences flanking the pesticin determinant were highly homologous to those flanking the colicin 10 determinant. It is proposed that through these highly homologous DNA sequences, genes encoding bacteriocins may be exchanged between plasmids by recombination. In the case of pesticin, recombination may have destroyed the lysis gene, of which only a rudimentary fragment exists on pPCP1.     

Translocation of jellyfish green fluorescent protein via the Tat system of Escherichia coli and change of its periplasmic localization in response to osmotic up-shock

The bacterial twin arginine translocation (Tat) pathway is capable of exporting cofactor-containing enzymes into the periplasm. To assess the capacity of the Tat pathway to export heterologous proteins and to gain information about the property of the periplasm, we fused the twin arginine signal peptide of the trimethylamine N-oxide reductase to the jellyfish green fluorescent protein (GFP). Unlike the Sec pathway, the Tat system successfully exported correctly folded GFP into the periplasm of Escherichia coli. Interestingly, GFP appeared as a halo in most cells and occasionally showed a polar localization in wild type strains. When subjected to a mild osmotic up-shock, GFP relocalized very quickly at the two poles of the cells. The conversion from the halo structure to a periplasmic gathering at particular locations was also observed with spherical cells of the DeltarodA-pbpA mutant or of the wild type strain treated with lysozyme. Therefore, the periplasm is not a uniform compartment and the polarization of GFP is unlikely to be caused by simple invagination of the cytoplasmic membrane at the poles. Moreover, the polar gathering of GFP is reversible; the reversion was accelerated by glucose and inhibited by azide and carbonyl cyanide m-chlorophenylhydrazone, indicating an active adaptation of the bacteria to the osmolarity in the medium. These results strongly suggest a relocalization of periplasmic substances in response to environmental changes. The polar area might be the preferential zone where bacteria sense the change in the environment.     

Autodisplay of the protease inhibitor aprotinin in Escherichia coli

The Kunitz type protease inhibitor aprotinin, containing three intramolecular disulfide bonds, was expressed on the surface of Escherichia coli by Autodisplay. For this purpose, the aprotinin gene was fused in-frame to the transporter domain encoding DNA region of the AIDA-I autotransporter protein. Culture of cells supplied with the artificial gene at reducing conditions resulted in the translocation of aprotinin to the cell surface. Correct folding of aprotinin was shown by high affinity to its target enzyme HLE. No surface translocation was detectable under non-reducing conditions, indicating the degradation of aprotinin in the periplasm. By the use of periplasmic-protease defective E. coli strains PW147, PW151, and PW152, under non-reducing conditions, significant amounts of aprotinin appeared in the periplasm but not at the surface. Our results indicate that aprotinin molecules, reaching stable conformation before transport across the outer membrane, are degraded in the periplasm due to proteolysis. In case folding can be prevented, i.e., by blocking disulfide bond formation in the periplasm, aprotinin is translocated and can adopt its active conformation at the cell surface.     

Structural insights into the T6SS effector protein Tse3 and the Tse3–Tsi3 complex from Pseudomonas aeruginosa reveal a calcium‐dependent membrane‐binding mechanism

The opportunistic pathogen Pseudomonas aeruginosa uses the type VI secretion system (T6SS) to deliver the muramidase Tse3 into the periplasm of rival bacteria to degrade their peptidoglycan (PG). Concomitantly, P. aeruginosa uses the periplasm‐localized immunity protein Tsi3 to prevent potential self‐intoxication caused by Tse3, and thus gains an edge over rival bacteria in fierce niche competition. Here, we report the crystal structures of Tse3 and the Tse3–Tsi3 complex. Tse3 contains an annexin repeat‐like fold at the N‐terminus and a G‐type lysozyme fold at the C‐terminus. One loop in the N‐terminal domain (Loop 12) and one helix (α9) from the C‐terminal domain together anchor Tse3 and the Tse3–Tsi3 complex to membrane in a calcium‐dependent manner in vitro, and this membrane‐binding ability is essential for Tse3's activity. In the C‐terminal domain, a Y‐shaped groove present on the surface likely serves as the PG binding site. Two calcium‐binding motifs are also observed in the groove and these are necessary for Tse3 activity. In the Tse3–Tsi3 structure, three loops of Tsi3 insert into the substrate‐binding groove of Tse3, and three calcium ions present at the interface of the complex are indispensable for the formation of the Tse3–Tsi3 complex.     

Exon grafting yields a "two active-site" lysozyme

The design of enzymes with enhanced stability and activity has long been a goal in protein engineering. We report a strategy to engineer an additional active site for human lysozyme, grafted the entire human lysozyme exon 2, which encodes the catalytically competent domain, into the gene at a position corresponding to an exposed loop region in the translated protein. Exon 2 grafting created a novel lysozyme with twice the activity of the wild type enzyme, equal activity came from each of the two active sites. We dissected the contributions of each active site using site-directed mutagenesis of the catalytic doublets of (E35A/D53A), circular dichroism, fluorescence spectra, and molecular modeling. Temperature and pH stability of the "two active-site" enzyme were similar to those of wild-type lysozyme. Thus, we provide a novel strategy for engineering the active site of enzymes.     

New Insights into the Assembly of Bacterial Secretins: STRUCTURAL STUDIES OF THE PERIPLASMIC DOMAIN OF XcpQ FROM PSEUDOMONAS AERUGINOSA*

The type II secretion system is a multiprotein assembly spanning the inner and outer membranes in Gram-negative bacteria. It is found in almost all pathogenic bacteria where it contributes to virulence, host tissue colonization, and infection. The exoproteins are secreted across the outer membrane via a large translocation channel, the secretin, which typically adopts a dodecameric structure. These secretin channels have large periplasmic N-terminal domains that reach out into the periplasm for communication with the inner membrane platform and with a pseudopilus structure that spans the periplasm. Here we report the crystal structure of the N-terminal periplasmic domain of the secretin XcpQ from Pseudomonas aeruginosa, revealing a two-lobe dimeric assembly featuring parallel subunits engaging in well defined interactions at the tips of each lobe. We have employed structure-based engineering of disulfide bridges and native mass spectrometry to show that the periplasmic domain of XcpQ dimerizes in a concentration-dependent manner. Validation of these insights in the context of cellular full-length XcpQ and further evaluation of the functionality of disulfide-linked XcpQ establishes that the basic oligomerization unit of XcpQ is a dimer. This is consistent with the notion that the dodecameric secretin assembles as a hexamer of dimers to ensure correct projection of the N-terminal domains into the periplasm. Therefore, our studies provide a key conceptual advancement in understanding the assembly principles and dynamic function of type II secretion system secretins and challenge recent studies reporting monomers as the basic subunit of the secretin oligomer.     

Transport of ferric-aerobactin into the periplasm and cytoplasm of Escherichia coli K12: role of envelope-associated proteins and effect of endogenous siderophores.

Purified [14C]aerobactin, supplied exogenously to non-growing bacteria, was translocated via the periplasm into the cytoplasm of Escherichia coli K12 strains expressing the aerobactin receptor protein IutA. No significant uptake was observed into either compartment of strains lacking the iutA gene or specifically defective in tonB. Uptake into both compartments was markedly reduced, but not abolished, in an exb mutant. Accumulation of [14C]aerobactin in the periplasm of fhuD, fhuB or fhuC mutant strains was not significantly lower than in the wild-type strain, but entry into the cytoplasm was greatly reduced in all cases. Uptake of aerobactin by strains wild-type for all transport functions occurred most efficiently in strains either lacking or specifically defective in the genetic determinants for aerobactin biosynthesis; significantly lower levels of exogenous 14C-labelled siderophore were observed in both compartments of strains producing aerobactin. Aerobactin-mediated 59Fe uptake, however, was not inhibited by the presence of endogenous aerobactin. Endogenous enterochelin did not affect aerobactin uptake.     

A helix-loop-helix peptide at the upper lip of the active site cleft of lysozyme confers potent antimicrobial activity with membrane permeabilization action

Recently, we have found that partially unfolded lysozyme exerts broad spectrum antimicrobial action in vitro against Gram-negative and Gram-positive bacteria independent of its catalytic activity. In parallel, an internal peptide (residues 98-112) of hen egg white lysozyme, obtained after digestion with clostripain, possessed broad spectrum antimicrobial action in vitro. This internal peptide is part of a helix-loop-helix domain (87-114 sequence of hen lysozyme) located at the upper lip of the active site cleft of lysozyme. The helix-loop-helix (HLH) structures are known motifs commonly found in membrane-active and DNA-binding proteins. To evaluate the contribution of the HLH peptide to the antimicrobial properties of lysozyme, the HLH sequence and its secondary structure derivatives of chicken and human lysozyme were synthesized and tested for antimicrobial activity against several bacterial strains. We found that the full HLH peptide of both chicken and human lysozymes was potently microbicidal against both Gram-positive and Gram-negative bacteria and the fungus Candida albicans. The N-terminal helix of HLH was specifically bactericidal to Gram-positive bacteria, whereas the C-terminal helix was bactericidal to all tested strains. Outer and inner membrane permeabilization studies, as well as measurements of transmembrane electrochemical potentials, provided evidence that HLH peptide and its C-terminal helix domain kill Gram-negative bacteria by crossing the outer membrane via self-promoted uptake and causing damage to the inner membrane through channel formation. The results are discussed in terms of proposed mechanisms for the catalytically independent antimicrobial activity of lysozyme that offer a new strategy for the design of potential antimicrobial drugs in the treatment of infectious diseases.     

Mapping functional domains of colicin M

Colicin M (Cma) lyses Escherichia coli cells by inhibiting murein biosynthesis through hydrolysis of the phosphate ester between C(55)-polyisoprenol and N-acetylmuramyl (MurNAc)-pentapeptide-GlcNAc in the periplasm. To identify Cma functional domains, we isolated 54 point mutants and small deletion mutants and examined their cytotoxicity levels. Activity and uptake mutants were distinguished by osmotic shock, which transfers Cma into the periplasm independent of the specific FhuA receptor and the Ton system. Deletion of the hydrophobic helix α1, which extends from the compact Cma structure, abolished interference with the antibiotic albomycin, which is transported across the outer membrane by the same system as Cma, thereby identifying α1 as the Cma site that binds to FhuA. Deletion of the C-terminal Lys-Arg strongly reduced Cma translocation across the outer membrane after binding to FhuA. Conversion of Asp226 to Glu, Asn, or Ala inactivated Cma. Asp226 is exposed at the Cma surface and is surrounded by Asp225, Asp229, His235, Tyr228, and Arg236; replacement of each with alanine inactivated Cma. We propose that Asp226 directly participates in phosphate ester hydrolysis and that the surrounding residues contribute to the active site. These residues are strongly conserved in Cma-like proteins of other species. Replacement of other conserved residues with alanine inactivated Cma; these mutations probably altered the Cma structure, as particularly apparent for mutants in the unique open β-barrel of Cma, which were isolated in lower yields. Our results identify regions in Cma responsible for uptake and activity and support the concept of a three-domain arrangement of Cma.     

Inactivation of gram-negative bacteria by lysozyme, denatured lysozyme, and lysozyme-derived peptides under high hydrostatic pressure

We have studied the inactivation of six gram-negative bacteria (Escherichia coli, Pseudomonas fluorescens, Salmonella enterica serovar Typhimurium, Salmonella enteritidis, Shigella sonnei, and Shigella flexneri) by high hydrostatic pressure treatment in the presence of hen egg-white lysozyme, partially or completely denatured lysozyme, or a synthetic cationic peptide derived from either hen egg white or coliphage T4 lysozyme. None of these compounds had a bactericidal or bacteriostatic effect on any of the tested bacteria at atmospheric pressure. Under high pressure, all bacteria except both Salmonella species showed higher inactivation in the presence of 100 microg of lysozyme/ml than without this additive, indicating that pressure sensitized the bacteria to lysozyme. This extra inactivation by lysozyme was accompanied by the formation of spheroplasts. Complete knockout of the muramidase enzymatic activity of lysozyme by heat treatment fully eliminated its bactericidal effect under pressure, but partially denatured lysozyme was still active against some bacteria. Contrary to some recent reports, these results indicate that enzymatic activity is indispensable for the antimicrobial activity of lysozyme. However, partial heat denaturation extended the activity spectrum of lysozyme under pressure to serovar Typhimurium, suggesting enhanced uptake of partially denatured lysozyme through the serovar Typhimurium outer membrane. All test bacteria were sensitized by high pressure to a peptide corresponding to amino acid residues 96 to 116 of hen egg white, and all except E. coli and P. fluorescens were sensitized by high pressure to a peptide corresponding to amino acid residues 143 to 155 of T4 lysozyme. Since they are not enzymatically active, these peptides probably have a different mechanism of action than all lysozyme polypeptides.     

Disulfide-dependent folding and export of Escherichia coli DsbC

DsbC, a member of the Dsb family in the periplasm of Gram-negative bacteria, is not only a disulfide isomerase but also a chaperone. Five DsbC mutants with Cys in the active site sequence of Cys(98)-Gly-Tyr-Cys(101) and the nonactive site disulfide Cys(141)-Cys(163) replaced by Ser have been studied. The results show that the active site Cys residues are necessary for enzyme activities but not required for chaperone activity, while the lack of the nonactive site disulfide results in a decreased chaperone activity in assisting the reactivation of denatured d-glyceraldehyde-3-phosphate dehydrogenase but has no effect on enzyme activities. Wild-type DsbC was overexpressed and correctly processed as a soluble periplasmic protein. Mutation in one of these Cys residues results in aggregation or extracellular/membrane locations, but does not affect the proper processing. DsbC mutated in either Cys residue of nonactive site disulfide shows higher sensitivity to unfolding by guanidine hydrochloride and slower refolding compared with wild-type DsbC and the active site Cys mutants. The above results provide experimental evidence for structural role of the nonactive site disulfide in folding and biological activities of DsbC.     

Functional and Structural Characterization of PaeM,a Colicin M-like Bacteriocin Produced by Pseudomonas aeruginosa

Colicin M (ColM) is the only enzymatic colicin reported to date that inhibits cell wall peptidoglycan biosynthesis. It catalyzes the specific degradation of the lipid intermediates involved in this pathway, thereby provoking lysis of susceptible Escherichia coli cells. A gene encoding a homologue of ColM was detected within the exoU-containing genomic island A carried by certain pathogenic Pseudomonas aeruginosa strains. This bacteriocin (pyocin) that we have named PaeM was crystallized, and its structure with and without an Mg²⁺ ion bound was solved. In parallel, site-directed mutagenesis of conserved PaeM residues from the C-terminal domain was performed, confirming their essentiality for the protein activity both in vitro (lipid II-degrading activity) and in vivo (cytotoxicity against a susceptible P. aeruginosa strain). Although PaeM is structurally similar to ColM, the conformation of their active sites differs radically; in PaeM, residues essential for enzymatic activity and cytotoxicity converge toward a same pocket, whereas in ColM they are spread along a particularly elongated active site. We have also isolated a minimal domain corresponding to the C-terminal half of the PaeM protein and exhibiting a 70-fold higher enzymatic activity as compared with the full-length protein. This isolated domain of the PaeM bacteriocin was further shown to kill E. coli cells when addressed to the periplasm of these bacteria.     

Activators of AMP-activated protein kinase enhance GLUT4 translocation and its glucose transport activity in 3T3-L1 adipocytes

To determine whether the increase in glucose uptake following AMP-activated protein kinase (AMPK) activation in adipocytes is mediated by accelerated GLUT4 translocation into plasma membrane, we constructed a chimera between GLUT4 and enhanced green fluorescent protein (GLUT4-eGFP) and transferred its cDNA into the nucleus of 3T3-L1 adipocytes. Then, the dynamics of GLUT4-eGFP translocation were visualized in living cells by means of laser scanning confocal microscopy. It was revealed that the stimulation with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) and 2,4-dinitrophenol (DNP), known activators of AMPK, promptly accelerates its translocation within 4 min, as was found in the case of insulin stimulation. The insulin-induced GLUT4 translocation was markedly inhibited after addition of wortmannin (P < 0.01). However, the GLUT4 translocation through AMPK activators AICAR and DNP was not affected by wortmannin. Insulin- and AMPK-activated translocation of GLUT4 was not inhibited by SB-203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK). Glucose uptake was significantly increased after addition of AMPK activators AICAR and DNP (P < 0.05). AMPK- and insulin-stimulated glucose uptake were similarly suppressed by wortmannin (P < 0.05-0.01). In addition, SB-203580 also significantly prevented the enhancement of glucose uptake induced by AMPK and insulin (P < 0.05). These results suggest that AMPK-activated GLUT4 translocation in 3T3-L1 adipocytes is mediated through the insulin-signaling pathway distal to the site of activated phosphatidylinositol 3-kinase or through a signaling system distinct from that activated by insulin. On the other hand, the increase of glucose uptake dependent on AMPK activators AICAR and DNP would be additionally due to enhancement of the intrinsic activity in translocated GLUT4 protein, possibly through a p38 MAPK-dependent mechanism.     

Structural and kinetic insights reveal that the amino acid pair Gln-228/Asn-254 modulates the transfructosylating specificity of Schwanniomyces occidentalis β-fructofuranosidase, an enzyme that produces prebiotics

Schwanniomyces occidentalis β-fructofuranosidase (Ffase) is a GH32 dimeric enzyme that releases fructose from the nonreducing end of various oligosaccharides and essential storage fructans such as inulin. It also catalyzes the transfer of a fructosyl unit to an acceptor producing 6-kestose and 1-kestose, prebiotics that stimulate the growth of bacteria beneficial for human health. We report here the crystal structure of inactivated Ffase complexed with fructosylnystose and inulin, which shows the intricate net of interactions keeping the substrate tightly bound at the active site. Up to five subsites were observed, the sugar unit located at subsite +3 being recognized by interaction with the β-sandwich domain of the adjacent subunit within the dimer. This explains the high activity observed against long substrates, giving the first experimental evidence of the direct role of a GH32 β-sandwich domain in substrate binding. Crucial residues were mutated and their hydrolase/transferase (H/T) activities were fully characterized, showing the involvement of the Gln-228/Asn-254 pair in modulating the H/T ratio and the type β(2-1)/β(2-6) linkage formation. We generated Ffase mutants with new transferase activity; among them, Q228V gives almost specifically 6-kestose, whereas N254T produces a broader spectrum product including also neokestose. A model for the mechanism of the Ffase transfructosylation reaction is proposed. The results contribute to an understanding of the molecular basis regulating specificity among GH-J clan members, which represent an interesting target for rational design of enzymes, showing redesigned activities to produce tailor-made fructooligosaccharides.     

Relocating the active site of activated protein C eliminates the need for its protein S cofactor. A fluorescence resonance energy transfer study

The effect of replacing the gamma-carboxyglutamic acid domain of activated protein C (APC) with that of prothrombin on the topography of the membrane-bound enzyme was examined using fluorescence resonance energy transfer. The average distance of closest approach (assuming kappa2 = 2/3) between a fluorescein in the active site of the chimera and octadecylrhodamine at the membrane surface was 89 A, compared with 94 A for wild-type APC. The gamma-carboxyglutamic acid domain substitution therefore lowered and/or reoriented the active site, repositioning it close to the 84 A observed for the APC. protein S complex. Protein S enhances wild-type APC cleavage of factor Va at Arg306, but the inactivation rate of factor Va Leiden by the chimera alone is essentially equal to that by wild-type APC plus protein S. These data suggest that the activities of the chimera and of the APC.protein S complex are equivalent because the active site of the chimeric protein is already positioned near the optimal location above the membrane surface to cleave Arg306. Thus, one mechanism by which protein S regulates APC activity is by relocating its active site to the proper position above the membrane surface to optimize factor Va cleavage.     

Probing secretion and translocation of a β-autotransporter using a reporter single-chain Fv as a cognate passenger domain

The mechanism of protein secretion mediated by the beta-domain of the Neisseria gonorrhoeae IgA protease, a paradigm of a family of secreted polypeptides of Gram-negative bacteria called autotransporters, has been examined using a single-chain antibody (scFv) as a reporter passenger domain to monitor the translocation process. Fusion of a scFv to the beta-module of the IgA protease allowed us to investigate the passage of the chimeric protein through the periplasm, its insertion into the outer membrane and the movement of the N-terminal moiety towards the cell surface. As the binding activity of the scFv to its target antigen is entirely dependent on the formation of disulphide bonds, the relationship between secretion, folding and formation of S-S bridges could be analysed in detail. In contrast to the current notion that only an unfolded N-passenger domain can be translocated through the beta-domain, our results show that the scFv is able to pass through the outer membrane, albeit at a threefold reduced level, in an active conformation with its disulphide bonds preformed in the periplasm through the action of the DsbA product. These data call for a re-evaluation of the prevailing model for secretion of the N-domain of autotransporters.     

YidC is involved in the biogenesis of the secreted autotransporter hemoglobin protease

Autotransporters (ATs) constitute an important family of virulence factors secreted by Gram-negative bacteria. Following their translocation across the inner membrane (IM), ATs temporarily reside in the periplasmic space after which they are secreted into the extracellular environment. Previous studies have shown that the AT hemoglobin protease (Hbp) of Escherichia coli requires a functional signal recognition particle pathway and Sec translocon for optimal targeting to and translocation across the IM. Here, we analyzed the mode of IM translocation of Hbp in more detail. Using site-directed photocross-linking, we found that the Hbp signal peptide is adjacent to YidC early during biogenesis. Notably, YidC is in part associated with the Sec translocon but has until now primarily been implicated in the biogenesis of IM proteins. In vivo, YidC appeared critical for the biogenesis of the ATs Hbp and EspC. For Hbp, depletion of YidC resulted in the formation of secretion-incompetent intermediates that were sensitive to degradation by the periplasmic protease DegP, indicating that YidC activity affects Hbp biogenesis at a late stage, after translocation across the IM. This is the first demonstration of a role for YidC in the biogenesis of an extracellular protein. We propose that YidC is required for maintenance of the translocation-competent state of certain ATs in the periplasm. The large periplasmic domain of YidC is not critical for this novel functionality as it can be deleted without affecting Hbp biogenesis.     

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.