PP1 regulatory subunit NIPP1 regulates transcription of E2F1 target genes following DNA damage.

DNA damage induces transcriptional repression of E2F1 target genes and a reduction in histone H3-Thr11 phosphorylation (H3-pThr11 ) at E2F1 target gene promoters. Dephosphorylation of H3-pThr11 is partly mediated by Chk1 kinase and protein phosphatase 1γ (PP1γ) phosphatase. Here, we isolated NIPP1 as a regulator of PP1γ-mediated H3-pThr11 by surveying nearly 200 PP1 interactor proteins. We found that NIPP1 inhibits PP1γ-mediated dephosphorylation of H3-pThr11 both in vivo and in vitro. By generating NIPP1-depleted cells, we showed that NIPP1 is required for cell proliferation and the expression of E2F1 target genes. Upon DNA damage, activated protein kinase A (PKA) phosphorylated the NIPP1-Ser199 residue, adjacent to the PP1 binding motif (RVxF), and triggered the dissociation of NIPP1 from PP1γ, leading to the activation of PP1γ. Furthermore, the inhibition of PKA activity led to the activation of E2F target genes. Statistical analysis confirmed that the expression of NIPP1 was positively correlated with E2F target genes. Taken together, these findings demonstrate that the PP1 regulatory subunit NIPP1 modulates E2F1 target genes by linking PKA and PP1γ during DNA damage.

Purification and characterization of a phospholipid-hydrolyzing phosphoesterase produced by Pediococcus acidilactici isolated from Gouda cheese.

Lipolysis occurs during ripening of dairy products as a result of esterase or lipase activity. Lactic acid bacteria (LAB) are considered to be weakly lipolytic bacteria compared with other species. In cheeses with extended ripening periods, lipolytic LAB may have several advantages. Pediococcus acidilactici is a LAB frequently found in fermented dairy products, but no previous reports exist on their production of esterases or lipases. Our interest in the relationship of LAB and enzymatic characterization is due to the multiple reports of the benefits of LAB in the gut microbiome, particularly at the intestinal membrane. Pediococci have been characterized as probiotic and especially active in membrane interactions. The aim of this project was to purify, characterize, and identify the phosphoesterase produced by P. acidilactici originally isolated from Gouda cheese and determine its phospholipid (PL) hydrolysis profile, with a focus on increased absorption of these compounds in the human gut. Native zymograms were performed to identify a protein with lipolytic activity in the intracellular fraction of P. acidilactici. The enzyme was purified via size-exclusion HPLC, concentrated via ultrafiltration, and identified using sequence analysis in liquid chromatography (LC)-MS/MS. The purified fraction was subjected to biochemical characterization as a function of pH, temperature, ion concentration, hydrolysis of different substrates, and PL. A single protein with a molecular weight of 86 kDa and esterase activity was detected by zymography. Analysis of the LC-MS/MS results identified a putative metallophosphoesterase with a calculated molecular weight of 45.5 kDa, suggesting that this protein is active as a homodimer. The pure protein showed an optimal activity between pH 8.0 to 9.0. The optimal temperature for activity was 37°C, and the enzyme lost 15% of activity after incubation at 90°C for 1 h. This enzyme showed activity on short-chain fatty acids and exhibited high hydrolysis of phosphatidylinositol. It also hydrolyzed phosphatidylserine, phosphatidylcholine, and sphingomyelin. Phosphatidylethanolamine was hydrolyzed but with less efficiency. The characteristics and lipolytic actions exerted by this protein obtained from LAB hold promise for a potential strain of esterase or lipase that may exert human health benefits through increased digestibility and absorption of nutrients found in dairy products.

Biochemical characterization of the phosphatase domain of the tumor suppressor PH domain leucine-rich repeat protein phosphatase.

PH domain leucine-rich repeat protein phosphatase (PHLPP) directly dephosphorylates and inactivates Akt and protein kinase C and is therefore a prime target for pharmacological intervention of two key signaling pathways, the phosphatidylinositol 3-kinase and diacylglycerol signaling pathways. Here we report on the first biochemical characterization of the phosphatase domain of a PHLPP family member. The human PHLPP1 and PHLPP2 phosphatase domains were expressed and purified from bacteria or insect cells and their activities compared to that of full-length proteins immunoprecipitated from mammalian cells. Biochemical analyses reveal that the PHLPP phosphatase domain effectively dephosphorylates synthetic and peptidic substrates, that its activity is modulated by metals and lipophilic compounds, and that it has relatively high thermal stability. Mutational analysis of PHLPP2 reveals an unusual active site architecture compared to the canonical architecture of PP2C phosphatases and identifies key acidic residues (Asp 806, Glu 989, and Asp 1024) and bulky aromatic residues (Phe 783 and Phe 808) whose mutation impairs activity. Consistent with a unique active site architecture, we identify inhibitors that discriminate between PHLPP2 and PP2Cα. These data establish PHLPP as a member of the PP2C family of phosphatases with a unique active site architecture.

A serine/threonine phosphatase encoded by MG_207 of Mycoplasma genitalium is critical for its virulence.

BACKGROUND: Bacterial signal transduction systems like two component system (TCS) and Serine/Threonine kinase (STK) and Serine/Threonine phosphatase (STP) play important roles in the virulence and pathogenesis of bacterial pathogens. Mycoplasma genitalium, a mollicute that causes the urogenital diseases urethritis and cervicitis in men and women, respectively, is a pathogen which lacks TCS but possesses STK/STP. In this study, we investigated the biochemical and virulence properties of an STP protein encoded by the gene MG_207 of this species. RESULTS: We overexpressed MG207 in Escherichia coli overexpression system as a recombinant His10MG207 protein and purified it with affinity chromatography. This recombinant protein readily hydrolyzed the substrate p-nitrophenyl phosphate (pNPP) in a dose-dependent manner. Additional studies using synthetic peptides as substrates revealed that the recombinant protein was able to hydrolyze the threonine phosphate. Further, a transposon insertion mutant strain of M. genitalium (TIM207) that lacks the protein MG207 showed differentially phosphorylated proteins when compared to the wild type G37 strain. Mass spectrometry revealed that some of the key proteins differentially phosphorylated in TIM207 strain were putative cytoskeletal protein encoded by the gene MG_328 and pyruvate dehydrogenase E1 α chain encoded by the gene MG_274. In addition, TIM207 was noticed to be less cytotoxic to HeLa cells and this correlated with the production of less hydrogen peroxide by this strain. This strain was also less efficient in inducing the differentiation of THP-1 cell line as compared to wild type M. genitalium. CONCLUSIONS: The results of the study suggest that MG207 is an important signaling protein of M. genitalium and its presence may be crucial for the virulence of this species.

Mass spectrometry approaches to study mammalian kinase and phosphatase associated proteins.

Reversible phosphorylation events regulate critical aspects of cellular biology by affecting protein conformation, cellular localization, enzymatic activity and associations with interaction partners. Kinases and phosphatases interact not only with their substrates but also with regulatory subunits and other proteins, including scaffolds. In recent years, affinity purification coupled to mass spectrometry (AP-MS) has proven to be a powerful tool to identify protein-protein interactions (PPIs) involving kinases and phosphatases. In this review we outline general considerations for successful AP-MS, and describe strategies that we have used to characterize the interactions of kinases and phosphatases in human cells.

Two ancient bacterial-like PPP family phosphatases from Arabidopsis are highly conserved plant proteins that possess unique properties.

Protein phosphorylation, catalyzed by the opposing actions of protein kinases and phosphatases, is a cornerstone of cellular signaling and regulation. Since their discovery, protein phosphatases have emerged as highly regulated enzymes with specificity that rivals their counteracting kinase partners. However, despite years of focused characterization in mammalian and yeast systems, many protein phosphatases in plants remain poorly or incompletely characterized. Here, we describe a bioinformatic, biochemical, and cellular examination of an ancient, Bacterial-like subclass of the phosphoprotein phosphatase (PPP) family designated the Shewanella-like protein phosphatases (SLP phosphatases). The SLP phosphatase subcluster is highly conserved in all plants, mosses, and green algae, with members also found in select fungi, protists, and bacteria. As in other plant species, the nucleus-encoded Arabidopsis (Arabidopsis thaliana) SLP phosphatases (AtSLP1 and AtSLP2) lack genetic redundancy and phylogenetically cluster into two distinct groups that maintain different subcellular localizations, with SLP1 being chloroplastic and SLP2 being cytosolic. Using heterologously expressed and purified protein, the enzymatic properties of both AtSLP1 and AtSLP2 were examined, revealing unique metal cation preferences in addition to a complete insensitivity to the classic serine/threonine PPP protein phosphatase inhibitors okadaic acid and microcystin. The unique properties and high conservation of the plant SLP phosphatases, coupled to their exclusion from animals, red algae, cyanobacteria, archaea, and most bacteria, render understanding the function(s) of this new subclass of PPP family protein phosphatases of particular interest.

Chronophin, a novel HAD-type serine protein phosphatase, regulates cofilin-dependent actin dynamics.

Cofilin is a key regulator of actin cytoskeletal dynamics whose activity is controlled by phosphorylation of a single serine residue. We report the biochemical isolation of chronophin (CIN), a unique cofilin-activating phosphatase of the haloacid dehalogenase (HAD) superfamily. CIN directly dephosphorylates cofilin with high specificity and colocalizes with cofilin in motile and dividing cells. Loss of CIN activity blocks phosphocycling of cofilin, stabilizes F-actin structures and causes massive cell division defects. Our findings identify a physiological phospho-serine protein substrate for a mammalian HAD-type phosphatase and demonstrate that CIN is an important novel regulator of cofilin-mediated actin reorganization.

Co-expression of protein phosphatases in insect cells affects phosphorylation status and expression levels of proteins.

The activity of kinases is regulated by phosphorylation on Ser, Thr or Tyr residues within the activation loop. The ability to produce these enzymes recombinantly with a specific phosphorylation status is essential in order to understand structure and function. In this paper we describe a screening approach to co-express different phosphatases together with a kinase in the baculovirus expression system. This enabled the testing of different phosphatases as well as different levels of both phosphatase and kinase by varying the multiplicity of infection (MOI) of the different baculoviruses. This approach translated well to a larger scale. An unexpected observation was that co-expression of the phosphatase could have profound effects on expression levels even of heterologous target proteins that would not be a substrate for the phosphatase. This was most apparent with lambda phosphatase, an enzyme that removes phosphorylation from Ser and Thr residues, where expression was almost completely abolished for all proteins, even at modest MOIs. The effect of lambda phosphatase was observed irrespective of whether co-expression was from two separate baculoviruses or from two genes on the same vector. The effect was shown to be due, in part at least, to a decrease in transcription.

Characterization of two putative protein phosphatase genes and their involvement in phosphorus efficiency in Phaseolus vulgaris.

Protein dephosphorylation mediated by protein phosphatases plays a major role in signal transduction of plant responses to environmental stresses. In this study, two putative protein phosphatases, PvPS2:1 and PvPS2:2 were identified and characterized in bean (Phaseolus vulgaris). The two PvPS2 members were found to be localized to the plasma membrane and the nucleus by transient expression of PvPS2:GFP in onion epidermal cells. Transcripts of the two PvPS2 genes were significantly increased by phosphate (P(i) ) starvation in the two bean genotypes, G19833 (a P-efficient genotype) and DOR364 (a P-inefficient genotype). However, G19833 exhibited higher PvPS2:1 expression levels than DOR364 in both leaves and roots during P(i) starvation. Increased transcription of PvPS2:1 in response to P(i) starvation was further verified through histochemical analysis of PvPS2:1 promoter fusion ß-glucuronidase (GUS) in transgenic Arabidopsis plants. Analysis of PvPS2:1 overexpression lines in bean hairy roots and Arabidopsis showed that PvS2:1 was involved in root growth and P accumulation. Furthermore, expression levels of two P(i) starvation responsive genes were upregulated and the APase activities were enhanced in the overexpressing PvPS2:1 Arabidopsis lines. Taken together, our results strongly suggested that PvPS2:1 positively regulated plant responses to P(i) starvation, and could be further targeted as a candidate gene to improve crop P efficiency.

Structure of human dual-specificity phosphatase 27 at 2.38†Šresolution.

There are over 100 genes in the human genome that encode protein tyrosine phosphatases (PTPs) and approximately 60 of these are classified as dual-specificity phosphatases (DUSPs). Although many dual-specificity phosphatases are still not well characterized, novel functions have been discovered for some of them that have led to new insights into a variety of biological processes and the molecular basis for certain diseases. Indeed, as the functions of DUSPs continue to be elucidated, a growing number of them are emerging as potential therapeutic targets for diseases such as cancer, diabetes and inflammatory disorders. Here, the overexpression, purification and structure determination of DUSP27 at 2.38 Šresolution are presented.

Heat-induced chaperone activity of serine/threonine protein phosphatase 5 enhances thermotolerance in Arabidopsis thaliana.

• This study reports that Arabidopsis thaliana protein serine/threonine phosphatase 5 (AtPP5) plays a pivotal role in heat stress resistance. A high-molecular-weight (HMW) form of AtPP5 was isolated from heat-treated A. thaliana suspension cells. AtPP5 performs multiple functions, acting as a protein phosphatase, foldase chaperone, and holdase chaperone. The enzymatic activities of this versatile protein are closely associated with its oligomeric status, ranging from low oligomeric protein species to HMW complexes. • The phosphatase and foldase chaperone functions of AtPP5 are associated primarily with the low-molecular-weight (LMW) form, whereas the HMW form exhibits holdase chaperone activity. Transgenic over-expression of AtPP5 conferred enhanced heat shock resistance to wild-type A. thaliana and a T-DNA insertion knock-out mutant was defective in acquired thermotolerance. A recombinant phosphatase mutant (H290N) showed markedly increased holdase chaperone activity. • In addition, enhanced thermotolerance was observed in transgenic plants over-expressing H290N, which suggests that the holdase chaperone activity of AtPP5 is primarily responsible for AtPP5-mediated thermotolerance. • Collectively, the results from this study provide the first evidence that AtPP5 performs multiple enzymatic activities that are mediated by conformational changes induced by heat-shock stress.

PP4 is a gamma H2AX phosphatase required for recovery from the DNA damage checkpoint.

Phosphorylation of histone H2AX on Ser 139 (gammaH2AX) is one of the earliest events in the response to DNA double-strand breaks; however, the subsequent removal of gammaH2AX from chromatin is less understood, despite being a process tightly coordinated with DNA repair. Previous studies in yeast have identified the Pph3 phosphatase (the PP4C orthologue) as important for the dephosphorylation of gammaH2AX. By contrast, work in human cells attributed this activity to PP2A. Here, we report that PP4 contributes to the dephosphorylation of gammaH2AX, both at the sites of DNA damage and in undamaged chromatin in human cells, independently of a role in DNA repair. Furthermore, depletion of PP4C results in a prolonged checkpoint arrest, most likely owing to the persistence of mediator of DNA damage checkpoint 1 (MDC1) at the sites of DNA lesions. Taken together, these results indicate that PP4 is an evolutionarily conserved gammaH2AX phosphatase.

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) is indispensable for normal embryogenesis in zebrafish, Danio rerio.

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) dephosphorylates and regulates multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs). However, the biological functions of this enzyme have not been clarified in vivo. To investigate the biological significance of CaMKP during zebrafish embryogenesis, we cloned and characterized zebrafish CaMKP (zCaMKP). The isolated cDNA clone possessed an open reading frame of 1272bp encoding 424 amino acids and shared 47% and 48% amino acid identity with rat and human CaMKP, respectively. Interestingly, zCaMKP lacks the Glu cluster corresponding to residues 101-109 in the rat enzyme, and was not activated by polycations such as poly-l-lysine. The recombinant zCaMKP required Mg(2+) rather than Mn(2+) for activity. Furthermore, zCaMKP dephosphorylated CaMKIV but not phosphorylase a, alpha-casein, or extracellular signal-regulating kinase (ERK), suggesting that the enzyme regulates Ca(2+) signaling pathways in zebrafish. Cotransfection of zCaMKP with mammalian CaMKI significantly decreased phospho-CaMKI in ionomycin-stimulated 293T cells. During embryogenesis, the expression of zCaMKP increased gradually after 48h post-fertilization, as demonstrated by Western blotting using an anti-zCaMKP antibody. The knockdown of the zCaMKP gene with morpholino-based antisense oligonucleotides resulted in an increased incidence of embryos with severe morphological and cellular abnormalities, i.e., a significant increase in the number of round-shaped embryos and apoptotic cells in the whole body. A marked decrease in zCaMKP expression was observed in the antisense- but not control oligo-injected embryos. Embryonic death was rescued by coinjection with recombinant rat CaMKP but not with phosphatase-dead mutant (D194A). These results clearly show the significance of zCaMKP during zebrafish embryogenesis.

Myosin light chain kinase and myosin light chain phosphatase from Dictyostelium: effects of reversible phosphorylation on myosin structure and function.

We have partially purified myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) from Dictyostelium discoideum. MLCK was purified 4,700-fold with a yield of approximately 1 mg from 350 g of cells. The enzyme is very acidic as suggested by its tight binding to DEAE. Dictyostelium MLCK has an apparent native molecular mass on HPLC G3000SW of approximately 30,000 D. Mg2+ is required for enzyme activity. Ca2+ inhibits activity and this inhibition is not relieved by calmodulin. cAMP or cGMP have no effect on enzyme activity. Dictyostelium MLCK is very specific for the 18,000-D light chain of Dictyostelium myosin and does not phosphorylate the light chain of several other myosins tested. Myosin purified from log-phase amebas of Dictyostelium has approximately 0.3 mol Pi/mol 18,000-D light chain as assayed by glycerol-urea gel electrophoresis. Dictyostelium MLCK can phosphorylate this myosin to a stoichiometry approaching 1 mol Pi/mol 18,000-D light chain. MLCP, which was partially purified, selectively removes phosphate from the 18,000-D light chain but not from the heavy chain of Dictyostelium myosin. Phosphatase-treated Dictyostelium myosin has less than or equal to 0.01 mol Pi/mol 18,000-D light chain. Phosphatase-treated myosin could be rephosphorylated to greater than or equal to 0.96 mol Pi/mol 18,000-D light chain by incubation with MLCK and ATP. We found myosin thick filament assembly to be independent of the extent of 18,000-D light-chain phosphorylation when measured as a function of ionic strength. However, actin-activated Mg2+-ATPase activity of Dictyostelium myosin was found to be directly related to the extent of phosphorylation of the 18,000-D light chain. MLCK-treated myosin moved in an in vitro motility assay (Sheetz, M. P., and J. A. Spudich, 1983, Nature (Lond.), 305:31-35) at approximately 1.4 micron/s whereas phosphatase-treated myosin moved only slowly or not at all. The effects of phosphatase treatment on the movement were fully reversed by subsequent treatment with MLCK.

Identification of calmodulin-binding proteins in chicken embryo fibroblasts.

We recently reported the detection of multiple classes of calmodulin-binding proteins in subcellular fractions of chicken embryo fibroblasts by using a gel binding procedure (Van Eldik, L.J., and W.H. Burgess, 1983, J. Biol. Chem., 258:4539-4547). In this report we identify many of these calmodulin-binding proteins and provide further evidence for the existence of multiple classes of calmodulin-binding proteins based on the interaction of these proteins with calmodulin and other calcium-modulated proteins. The fact that, in some cases, the same calmodulin-binding protein can bind troponin C and S100 alpha suggests that similar functional domains may be present in these distinct calcium-modulated proteins. We also have used protocols based on purification steps for calmodulin-binding proteins and calmodulin-regulated activities from other systems, in conjunction with enzymatic assays and various immunological methods, to identify many of the calmodulin-binding proteins in chicken embryo fibroblasts. The identities of these proteins suggest in vivo roles for calmodulin in the regulation of cell shape and motility, cyclic nucleotide metabolism, and possibly nucleic acid and protein turnover in fibroblasts.

Protein phosphatase type-1, not type-2A, modulates actin microfilament integrity and myosin light chain phosphorylation in living nonmuscle cells.

Dynamic reorganization of the actin microfilament networks is dependent on the reversible phosphorylation of myosin light chain. To assess the potential role of protein phosphatases in this process in living nonmuscle cells, we have microinjected the purified type-1 and type-2A phosphatases into the cytoplasm of mammalian fibroblasts. Our studies reveal that elevating type-1 phosphatase levels led to the rapid (within 30 min) and fully reversible disassembly of the actin microfilament network as determined by immunofluorescence analysis. In contrast, microinjection of equivalent amounts of the purified type-2A phosphatase had no effect on actin microfilament organization. Metabolic labeling of cells after injection of purified phosphatases was used to analyze changes in protein phosphorylation. Concomitant with the disassembly of the actin microfilaments induced by type-1 phosphatase, there was an extensive dephosphorylation of myosin light chain. No such change was observed when cells were injected with type-2A phosphatase. In addition, after extraction of fibroblasts with Triton X-100, the type-1 phosphatase could be specifically localized by immunofluorescence to a fibrillar network of microfilaments. Furthermore, neutralizing type-1 phosphatase activity in vivo by microinjection of an affinity-purified antibody, prevented the reorganization of actin microfilaments that we had previously described following injection of cAMP-dependent protein kinase. These data support the notion that type 1 and type-2 phosphatases have distinct substrate specificity in living cells, and that type-1 phosphatase plays a predominant role in the dephosphorylation of myosin light chain and thus in the modulation of actin microfilament organization in vivo in intact nonmuscle cells.

Inhibition of a constitutive translation initiation factor 2alpha phosphatase, CReP, promotes survival of stressed cells.

Phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha) on serine 51 is effected by specific stress-activated protein kinases. eIF2alpha phosphorylation inhibits translation initiation promoting a cytoprotective gene expression program known as the integrated stress response (ISR). Stress-induced activation of GADD34 feeds back negatively on this pathway by promoting eIF2alpha dephosphorylation, however, GADD34 mutant cells retain significant eIF2alpha-directed phosphatase activity. We used a somatic cell genetic approach to identify a gene encoding a novel regulatory subunit of a constitutively active holophosphatase complex that dephosphorylates eIF2alpha. RNAi of this gene, which we named constitutive repressor of eIF2alpha phosphorylation (CReP, or PPP1R15B), repressed the constitutive eIF2alpha-directed phosphatase activity and activated the ISR. CReP RNAi strongly protected mammalian cells against oxidative stress, peroxynitrite stress, and more modestly against accumulation of malfolded proteins in the endoplasmic reticulum. These findings suggest that therapeutic inhibition of eIF2alpha dephosphorylation by targeting the CReP-protein-phosphatase-1 complex may be used to access the salubrious qualities of the ISR.

Characterization of the 110-kdalton actin-calmodulin-, and membrane-binding protein from microvilli of intestinal epithelial cells.

One of the major proteins of the chicken intestinal microvillus is a calmodulin-binding protein of 105-110 kdaltons which has been tentatively identified as the bridge linking the microvillar filament bundle laterally to the membrane. We have treated isolated, membrane-intact brush borders with ATP and obtained solubilization of the 110-kdalton protein, calmodulin (CM), myosin, and lesser amounts of several other cytoskeletal proteins. Electron micrographs of ATP-extracted brush borders showed loss of the linkers between the actin filament bundle and the microvillar membrane, with "ballooning" of the membrane away from the filament bundle, particularly at the tip end. In brush borders treated with calcium and trifluoperazine to solubilize CM, precise arrangement and morphology of lateral bridges was unperturbed, but ATP treatment would no longer solubilize the 110-kdalton protein. This result suggests that associated CM is necessary for the ATP-induced solubilization of the 110-kdalton protein. A 110-kdalton protein-CM complex, with 110-kdalton protein: CM ratios of 1:1-2, was partially purified from ATP-extracts of brush borders by a combination of gel filtration and hydroxylapatite chromatography. The 110-kdalton protein-CM complex is an irregular, elongated molecule that ranged in size from 5 X 8 nm to 8 X 14 nm, with a Stokes' radius of 6.1 nm. This 110-kdalton protein-CM complex exhibited no Mg++-ATPase activity and no detectable myosin light chain kinase activity. In co-sedimentation assays, the 110-kdalton protein-CM bound to F-actin in the absence but not the presence of ATP. Both the interaction of the complex with actin and the binding of CM to the 110-kdalton protein were calcium-independent. Negative stains of F-actin and 110-kdalton protein-CM in the absence of ATP showed loosely organized aggregates of actin with the 110-kdalton protein-CM complex coating the surface of the filaments. On the basis of our data, and in agreement with previous calculations (Matsudaira, P.T., and D.R. Burgess, 1979, J. Cell Biol. 83:667-673), we suggest that the lateral bridge of the microvillus is composed of a dimer of the 110-kdalton protein with four associated calmodulins.

A signaling complex of Ca2+-calmodulin-dependent protein kinase IV and protein phosphatase 2A.

Stimulation of T lymphocytes results in a rapid increase in intracellular calcium concentration ([Ca2+]i) that parallels the activation of Ca2+-calmodulin-dependent protein kinase IV (CaMKIV), a nuclear enzyme that can phosphorylate and activate the cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB). However, inactivation of CaMKIV occurs despite the sustained increase in [Ca2+]i that is required for T cell activation. A stable and stoichiometric complex of CaMKIV with protein serine-threonine phosphatase 2A (PP2A) was identified in which PP2A dephosphorylates CaMKIV and functions as a negative regulator of CaMKIV signaling. In Jurkat T cells, inhibition of PP2A activity by small t antigen enhanced activation of CREB-mediated transcription by CaMKIV. These findings reveal an intracellular signaling mechanism whereby a protein serine-threonine kinase (CaMKIV) is regulated by a tightly associated protein serine-threonine phosphatase (PP2A).

p38-MAPK induced dephosphorylation of alpha-tropomyosin is associated with depression of myocardial sarcomeric tension and ATPase activity.

Our objective in work presented here was to understand the mechanisms by which activated p38alpha MAPK depresses myocardial contractility. To test the hypothesis that activation of p38 MAPK directly influences sarcomeric function, we used transgenic mouse models with hearts in which p38 MAPK was constitutively turned on by an upstream activator (MKK6bE). These hearts demonstrated a significant depression in ejection fraction after induction of the transgene. We also studied hearts of mice expressing a dominant negative p38alpha MAPK. Simultaneous determination of tension and ATPase activity of detergent-skinned fiber bundles from left ventricular papillary muscle demonstrated a significant inhibition of both maximum tension and ATPase activity in the transgenic-MKK6bE hearts. Fibers from hearts expressing dominant negative p38alpha MAPK demonstrated no significant change in tension or ATPase activity. There were no significant changes in phosphorylation level of troponin-T3 and troponin-T4, or myosin light chain 2. However, compared with controls, there was a significant depression in levels of phosphorylation of alpha-tropomyosin and troponin I in fiber bundles from transgenic-MKK6bE hearts, but not from dominant negative p38alpha MAPK hearts. Our experiments also showed that p38alpha MAPK colocalizes with alpha-actinin at the Z-disc and complexes with protein phosphatases (PP2alpha, PP2beta). These data are the first to indicate that chronic activation of p38alpha MAPK directly depresses sarcomeric function in association with decreased phosphorylation of alpha-tropomyosin.