Dephosphorylation specificities of protein phosphatase for cardiac troponin I, troponin T, and sites within troponin T

Protein dephosphorylation by protein phosphatase 1 (PP1), acting in concert with protein kinase C (PKC) and protein kinase A (PKA), is a pivotal regulatory mechanism of protein phosphorylation. Isolated rat cardiac myofibrils phosphorylated by PKC/PKA and dephosphorylated by PP1 were used in determining dephosphorylation specificities, Ca(2+)-stimulated Mg(2+)ATPase activities, and Ca(2+) sensitivities. In reconstituted troponin (Tn) complex, PP1 displayed distinct substrate specificity in dephosphorylation of TnT preferentially to TnI, in vitro. In situ phosphorylation of cardiomyocytes with calyculin A, a protein phosphatase inhibitor, resulted in an increase in the phosphorylation stiochiometry of TnT (0.3 to 0.5 (67%)), TnI (2.6 to 3.6 (38%)), and MLC2 (0.4 to 1.7 (325%)). These results further confirmed that though MLC2 is the preferred target substrate for protein phosphatase in the thick filament, the Tn complex (TnI and TnT) from thin filament and C-protein in the thick filament are also protein phosphatase substrates. Our in vitro dephosphorylation experiments revealed that while PP1 differentially dephosphorylated within TnT at multiple sites, TnI was uniformly dephosphorylated. Phosphopeptide maps from the in vitro experiments show that TnT phosphopeptides at spots 4A and 4B are much more resistant to PP1 dephosphorylation than other TnT phosphopeptides. Mg(2+)ATPase assays of myofibrils phosphorylated by PKC/PKA and dephosphorylated by PP1 delineated that while PKC and PKA phosphorylation decreased the Ca(2+)-stimulated Mg(2+)ATPase activities, dephosphorylation antagonistically restored it. PKC and PKA phosphorylation decreased Ca(2+) sensitivity to 3.6 microM and 5.0 microM respectively. However, dephosphorylation restored the Mg(2+)ATPase activity of PKC (99%) and PKA (95%), along with the Ca(2+) sensitivities (3.3 microM and 3.0 microM, respectively).     

Phosphatidylinositol phosphate kinase type Igamma directly associates with and regulates Shp-1 tyrosine phosphatase

Tyrosine phosphorylation plays a critical role in many regulatory aspects of cellular signaling, and dephosphorylation of phosphotyrosine residues is crucial for termination of signals initiated by tyrosine kinases. Previous work has shown that the tyrosine kinase Src phosphorylates Tyr644 on phosphatidylinositol phosphate kinase type I (PIPKI) gamma661 in a focal adhesion kinase-dependent manner. Phosphorylation of this residue is essential for high affinity binding of PIPKI gamma661 to the focal adhesion protein talin and for targeting of PIPKI gamma661 to focal adhesions. A yeast two-hybrid screen performed with the C-terminal 178-amino acid tail of PIPKI gamma661 identified an interaction with the phosphatase domain of the tyrosine phosphatase Shp-1. The interaction between PIPKI gamma661 and Shp-1 was confirmed via co-immunoprecipitation from HEK293 cell lysates. In addition, Src-phosphorylated PIPKI gamma661 is a substrate for Shp-1, and Shp-1 modulates both the association between PIPKI gamma661 and talin and the targeting of PIPKI gamma661 to focal adhesions in mammalian cells. Finally, we showed that Shp-1 phosphatase activity is inhibited by the product of PIPKI gamma661, phosphatidylinositol 4,5-bisphosphate, in vitro. These combined results suggest a model in which the reciprocal actions of Src tyrosine kinase and Shp-1 tyrosine phosphatase dynamically regulate the association between PIPKI gamma661 and talin.     

Phosphorylation of TIMAP by glycogen synthase kinase-3beta activates its associated protein phosphatase 1

TIMAP (TGF-beta1 inhibited, membrane-associated protein) is a prenylated, endothelial cell-predominant protein phosphatase 1 (PP1c) regulatory subunit that localizes to the plasma membrane of filopodia. Here, we determined whether phosphorylation regulates TIMAP-associated PP1c function. Phosphorylation of TIMAP was observed in cells metabolically labeled with [32P]orthophosphate and was reduced by inhibitors of protein kinase A (PKA) and glycogen synthase kinase-3 (GSK-3). In cell-free assays, immunopurified TIMAP was phosphorylated by PKA and, after PKA priming, by GSK-3beta. Site-specific Ser to Ala substitution identified amino acid residues Ser333/Ser337 as the likely PKA/GSK-3beta phosphorylation site. Substitution of Ala for Val and Phe in the KVSF motif of TIMAP (TIMAPV64A/F66A) abolished PP1c binding and TIMAP-associated PP1c activity. TIMAPV64A/F66A was hyper-phosphorylated in cells, indicating that TIMAP-associated PP1c auto-dephosphorylates TIMAP. Constitutively active GSK-3beta stimulated phosphorylation of TIMAPV64A/F66A, but not wild-type TIMAP, suggesting that the PKA/GSK-3beta site may be subject to dephosphorylation by TIMAP-associated PP1c. Substitution of Asp or Glu for Ser at amino acid residues 333 and 337 to mimic phosphorylation reduced the PP1c association with TIMAP. Conversely, GSK-3 inhibitors augmented PP1c association with TIMAP-PP1c in cells. The 333/337 phosphomimic mutations also increased TIMAP-associated PP1c activity in vitro and against the non-integrin laminin receptor 1 in cells. Finally, TIMAP mutants with reduced PP1c activity strongly stimulated endothelial cell filopodia formation, an effect mimicked by the GSK-3 inhibitor LiCl. We conclude that TIMAP is a target for PKA-primed GSK-3beta-mediated phosphorylation. This phosphorylation controls TIMAP association and activity of PP1c, in turn regulating extension of filopodia in endothelial cells.     

Multiple interactions within the AKAP220 signaling complex contribute to protein phosphatase 1 regulation

The phosphorylation status of cellular proteins is controlled by the opposing actions of protein kinases and phosphatases. Compartmentalization of these enzymes is critical for spatial and temporal control of these phosphorylation/dephosphorylation events. We previously reported that a 220-kDa A-kinase anchoring protein (AKAP220) coordinates the location of the cAMP-dependent protein kinase (PKA) and the type 1 protein phosphatase catalytic subunit (PP1c) (Schillace, R. V., and Scott, J. D. (1999) Curr. Biol. 9, 321-324). We now demonstrate that an AKAP220 fragment is a competitive inhibitor of PP1c activity (K(i) = 2.9 +/- 0.7 micrometer). Mapping studies and activity measurements indicate that several protein-protein interactions act synergistically to inhibit PP1. A consensus targeting motif, between residues 1195 and 1198 (Lys-Val-Gln-Phe), binds but does not affect enzyme activity, whereas determinants between residues 1711 and 1901 inhibit the phosphatase. Analysis of truncated PP1c and chimeric PP1/2A catalytic subunits suggests that AKAP220 inhibits the phosphatase in a manner distinct from all known PP1 inhibitors and toxins. Intermolecular interactions within the AKAP220 signaling complex further contribute to PP1 inhibition as addition of the PKA regulatory subunit (RII) enhances phosphatase inhibition. These experiments indicate that regulation of PP1 activity by AKAP220 involves a complex network of intra- and intermolecular interactions.     

Coordinated control of endothelial nitric-oxide synthase phosphorylation by protein kinase C and the cAMP-dependent protein kinase

Endothelial nitric-oxide synthase (eNOS) is an important regulatory enzyme in the cardiovascular system catalyzing the production of NO from arginine. Multiple protein kinases including Akt/PKB, cAMP-dependent protein kinase (PKA), and the AMP-activated protein kinase (AMPK) activate eNOS by phosphorylating Ser-1177 in response to various stimuli. During VEGF signaling in endothelial cells, there is a transient increase in Ser-1177 phosphorylation coupled with a decrease in Thr-495 phosphorylation that reverses over 10 min. PKC signaling in endothelial cells inhibits eNOS activity by phosphorylating Thr-495 and dephosphorylating Ser-1177 whereas PKA signaling acts in reverse by increasing phosphorylation of Ser-1177 and dephosphorylation of Thr-495 to activate eNOS. Both phosphatases PP1 and PP2A are associated with eNOS. PP1 is responsible for dephosphorylation of Thr-495 based on its specificity for this site in both eNOS and the corresponding synthetic phosphopeptide whereas PP2A is responsible for dephosphorylation of Ser-1177. Treatment of endothelial cells with calyculin selectively blocks PKA-mediated dephosphorylation of Thr-495 whereas okadaic acid selectively blocks PKC-mediated dephosphorylation of Ser-1177. These results show that regulation of eNOS activity involves coordinated signaling through Ser-1177 and Thr-495 by multiple protein kinases and phosphatases.     

Insulin-induced beta-arrestin1 Ser-412 phosphorylation is a mechanism for desensitization of ERK activation by Galphai-coupled receptors

Beta-arrestin1 is an adapter/scaffold for many G protein-coupled receptors during mitogen-activated protein kinase signaling. Phosphorylation of beta-arrestin1 at position Ser-412 is a regulator of beta-arrestin1 function, and in the present study, we showed that insulin led to a time- and dose-dependent increase in beta-arrestin1 Ser-412 phosphorylation, which blocked isoproterenol- and lysophosphatidic acid-induced Ser-412 dephosphorylation and impaired ERK signaling by these G protein-coupled receptor ligands. Insulin treatment also led to accumulation of Ser-412-phosphorylated beta-arrestin1 at the insulin-like growth factor 1 receptor and prevented insulin-like growth factor 1/Src association. Insulin-induced Ser-412 phosphorylation was partially dependent on ERK as treatment with the MEK inhibitor PD98059 inhibited the insulin effect (62% reduction, p = 0.03). Inhibition of phosphatidylinositol 3-kinase by wortmannin did not have a significant effect (9% reduction, p = 0.41). We also found that the protein phosphatase 2A (PP2A) was in a molecular complex with beta-arrestin1 and that the PP2A inhibitor okadaic acid increased Ser-412 phosphorylation. Concomitant addition of insulin and okadaic acid did not produce an additive effect on Ser-412 phosphorylation, suggesting a common mechanism. Small t antigen specifically inhibited PP2A, and in HIRcB cells expressing small t antigen, beta-arrestin1 Ser-412 phosphorylation was increased, and insulin had no further effect. Insulin treatment caused increased beta-arrestin1 Ser-412 phosphorylation, which blocked mitogen-activated protein kinase signaling and internalization by beta-arrestin1-dependent receptors with no effect on beta-adrenergic receptor Gs-mediated cAMP production. These findings provide a new mechanism for insulin-induced desensitization of ERK activation by Galphai-coupled receptors.     

Protein phosphatase 2A is associated with class C L-type calcium channels (Cav1.2) and antagonizes channel phosphorylation by cAMP-dependent protein kinase

Phosphorylation by cAMP-dependent protein kinase (PKA) regulates a vast number of cellular functions. An important target for PKA in brain and heart is the class C L-type Ca(2+) channel (Ca(v)1.2). PKA phosphorylates serine 1928 in the central, pore-forming alpha(1C) subunit of this channel. Regulation of channel activity by PKA requires a proper balance between phosphorylation and dephosphorylation. For fast and specific signaling, PKA is recruited to this channel by an protein kinase A anchor protein (Davare, M. A., Dong, F., Rubin, C. S., and Hell, J. W. (1999) J. Biol. Chem. 274, 30280-30287). A phosphatase may be associated with the channel to effectively balance serine 1928 phosphorylation by channel-bound PKA. Dephosphorylation of this site is mediated by a serine/threonine phosphatase that is inhibited by okadaic acid and microcystin. We show that immunoprecipitation of the channel complex from rat brain results in coprecipitation of PP2A. Stoichiometric analysis indicates that about 80% of the channel complexes contain PP2A. PP2A directly and stably binds to the C-terminal 557 amino acids of alpha(1C). This interaction does not depend on serine 1928 phosphorylation and is not altered by PP2A catalytic site inhibitors. These results indicate that the PP2A-alpha(1C) interaction constitutively recruits PP2A to the channel complex rather than being a transient substrate-catalytic site interaction. Functional assays with the immunoisolated class C channel complex showed that channel-associated PP2A effectively reverses serine 1928 phosphorylation by endogenous PKA. Our findings demonstrate that both PKA and PP2A are integral components of the class C L-type Ca(2+) channel that determine the phosphorylation level of serine 1928 and thereby channel activity.     

Luteinizing hormone receptor activation in ovarian granulosa cells promotes protein kinase A-dependent dephosphorylation of microtubule-associated protein 2D

The actions of LH to induce ovulation and luteinization of preovulatory follicles are mediated principally by activation of cAMP-dependent protein kinase (PKA) in granulosa cells. PKA activity is targeted to specific locations in many cells by A kinase-anchoring proteins (AKAPs). We previously showed that FSH induces expression of microtubule-associated protein (MAP) 2D, an 80-kDa AKAP, in rat granulosa cells, and that MAP2D coimmunoprecipitates with PKA-regulatory subunits in these cells. Here we report a rapid and targeted dephosphorylation of MAP2D at Thr256/Thr259 after treatment with human chorionic gonadotropin, an LH receptor agonist. This event is mimicked by treatment with forskolin or a cAMP analog and is blocked by the PKA inhibitor myristoylated-PKI, indicating a role for cAMP and PKA signaling in phosphoregulation of granulosa cell MAP2D. Furthermore, we show that Thr256/Thr259 dephosphorylation is blocked by the protein phosphatase 2A (PP2A) inhibitor, okadaic acid, and demonstrate interactions between MAP2D and PP2A by coimmunoprecipitation and microcystin-agarose pull-down. We also show that MAP2D interacts with glycogen synthase kinase (GSK) 3beta and is phosphorylated at Thr256/Thr259 by this kinase in the basal state. Increased phosphorylation of GSK3beta at Ser9 and the PP2A B56delta subunit at Ser566 is observed after treatment with human chorionic gonadotropin and appears to result in LH receptor-mediated inhibition of GSK3beta and activation of PP2A, respectively. Taken together, these results show that the phosphorylation status of the AKAP MAP2D is acutely regulated by LH receptor-mediated modulation of kinase and phosphatase activities via PKA.     

The guanine-nucleotide-exchange factor P-Rex1 is activated by protein phosphatase 1α

P-Rex1 is a GEF (guanine-nucleotide-exchange factor) for the small G-protein Rac that is activated by PIP3 (phosphatidylinositol 3,4,5-trisphosphate) and Gβγ subunits and inhibited by PKA (protein kinase A). In the present study we show that PP1α (protein phosphatase 1α) binds P-Rex1 through an RVxF-type docking motif. PP1α activates P-Rex1 directly in vitro, both independently of and additively to PIP3 and Gβγ. PP1α also substantially activates P-Rex1 in vivo, both in basal and PDGF (platelet-derived growth factor)- or LPA (lysophosphatidic acid)-stimulated cells. The phosphatase activity of PP1α is required for P-Rex1 activation. PP1β, a close homologue of PP1α, is also able to activate P-Rex1, but less effectively. PP1α stimulates P-Rex1-mediated Rac-dependent changes in endothelial cell morphology. MS analysis of wild-type P-Rex1 and a PP1α-binding-deficient mutant revealed that endogenous PP1α dephosphorylates P-Rex1 on at least three residues, Ser834, Ser1001 and Ser1165. Site-directed mutagenesis of Ser1165 to alanine caused activation of P-Rex1 to a similar degree as did PP1α, confirming Ser1165 as a dephosphorylation site important in regulating P-Rex1 Rac-GEF activity. In summary, we have identified a novel mechanism for direct activation of P-Rex1 through PP1α-dependent dephosphorylation.     

Regulation of dopamine D-receptor activation in vivo by protein phosphatase 2B (calcineurin)

Mice lacking dopamine D2 receptors exhibit a significantly decreased agonist-promoted forebrain neocortical D1 receptor activation that occurs without changes in D1 receptor expression levels. This raises the possibility that, in brains of D2 mutants, a substantial portion of D1 receptors are uncoupled from their G protein, a phenomenon known as receptor desensitization. To test this, we examined D1-agonist-stimulated [35S]GTPgammaS binding (in the presence and absence of protein phosphatase inhibitors) and cAMP production (in the presence and absence of pertussis toxin) in forebrain neocortical tissues of wild-type mice and D2-receptor mutants. These studies revealed a decreased agonist-stimulated G-protein activation in D2 mutants. Moreover, whereas protein phosphatase 1/2A (PP1/2A) and 2B (PP2B) inhibitors decrease [35S]GTPgammaS binding in a concentration-dependent manner in wild type, they have either no (PP2B) or only partial (PP1/2A) effects in D2 mutants. Furthermore, for D2 mutants, immunoprecipitation experiments revealed increased basal and D1-agonist-stimulated phosphorylation of D1-receptor proteins at serine residues. Finally, D1 immunoprecipitates of both wild type and D2 mutants also contain protein kinase A (PKA) and PP2B immunoreactivities. In D2 mutants, however, the catalytic activity of the immunoprecipitated PP2B is abolished. These data indicate that neocortical D1 receptors are physically linked to PKA and PP2B and that the increased phosphorylation of D1 receptors in brains of D2 mutants is due to defective dephosphorylation of the receptor rather than increased kinase-mediated phosphorylation.     

Interaction and feedback regulation between STK15/BTAK/Aurora-A kinase and protein phosphatase 1 through mitotic cell division cycle

STK15 is an Aurora/Ipl-1 related serine/threonine kinase that is associated with centrosomes and induces aneuploidy when overexpressed in mammalian cells. It is well known that phosphorylation and dephosphorylation of kinases are important for regulation of their activity. But mechanisms by which STK15 activity is regulated have not been elucidated. We report that STK15 contains two functional binding sites for protein phosphatase type 1 (PP1), and the binding of these proteins is cell cycle-regulated peaking at mitosis. Activated STK15 at mitosis phosphorylates PP1 and inhibits PP1 activity in vitro. In vivo, PP1 activity co-immunoprecipitated with STK15 is also reduced. These data indicate that STK15 inhibits PP1 activity during mitosis. Also, PP1 is shown to dephosphorylate active STK15 and abolish its activity in vitro. Furthermore, we show that non-binding mutants of STK15 for PP1 are superphosphorylated, but their kinase activities are markedly reduced. Cells transfected with these non-binding mutants manifest aberrant chromosome alignment during mitosis. Our results suggest that a feedback regulation through phosphorylation/dephosphorylation events between STK15 kinase and PP1 phosphatase operates through the cell cycle. Deregulation of this balance may contribute to anomalous segregation of chromosomes during mitotic progression of cancer cells.     

Targeting of PKA, PKC and protein phosphatases to cellular microdomains

The intracellular responses to many distinct extracellular signals involve the direction of broad-based protein kinases and protein phosphatases to catalyse quite specific protein phosphorylation/dephosphorylation events. It is now clear that such specificity is often achieved through subcellular targeting of distinct pools of kinase or phosphatase towards particular substrates at specific subcellular locations. Given the dynamic nature of protein phosphorylation reactions, coordinated control of both kinase and phosphatases is often required and complexes formed by common scaffold or targeting proteins exist to direct both kinase and phosphatase to the same subcellular location. In many cases more than one kinase or phosphatase is required and binding proteins which target more than one kinase or phosphatase have now been identified. This review summarizes recent findings relating to the concept of targeting PKA, PKC and the major serine/threonine phosphatases, PP1, PP2A and PP2B, through the formation of multi-enzyme signalling complexes.     

3-phosphoinositide-dependent protein kinase 1, an Akt1 kinase, is involved in dephosphorylation of Thr-308 of Akt1 in Chinese hamster ovary cells

To investigate the role of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in the Akt1 phosphorylation state, wild-type (wt) PDK1 and its kinase dead (kd) mutant were expressed using an adenovirus gene transduction system in Chinese hamster ovary cells stably expressing insulin receptor. Immunoblotting using anti-phosphorylated Akt1 antibody revealed Thr-308 already to be maximally phosphorylated at 1 min but completely dephosphorylated at 5 min, with insulin stimulation, whereas insulin-induced Akt1 activation was maintained even after dephosphorylation of Thr-308. Overexpression of wt-PDK1 further increased insulin-stimulated phosphorylation of Thr-308, also followed by rapid dephosphorylation. The insulin-stimulated Akt1 activity was also enhanced by wt-PDK1 expression but was maintained even at 15 min. Thus, phosphorylation of Thr-308 is not essential for maintaining the Akt1 activity once it has been achieved. Interestingly, the insulin-stimulated phosphorylation state of Thr-308 was maintained even at 15 min in cells expressing kd-PDK1, suggesting that kd-PDK1 has a dominant negative effect on dephosphorylation of Thr-308 of Akt1. Calyculin A, an inhibitor of PP1 and PP2A, also prolonged the insulin-stimulated phosphorylation state of Thr-308. In addition, in vitro experiments revealed PP2A, but not PP1, to dephosphorylate completely Thr-308 of Akt1. These findings suggest that a novel pathway involving dephosphorylation of Akt1 at Thr-308 by a phosphatase, possibly PP2A, originally, identified as is regulated downstream from PDK1, an Akt1 kinase.     

Binding of protein phosphatase 2A to the L-type calcium channel Cav1.2 next to Ser1928, its main PKA site, is critical for Ser1928 dephosphorylation

The cAMP-dependent protein kinase (PKA) controls a large number of cellular functions. One critical PKA substrate in the brain and heart is the L-type Ca(2+) channel Ca(v)1.2, the activity of which is upregulated by PKA. The main PKA phosphorylation site is serine 1928 in the central pore forming alpha(1)1.2 subunit of Ca(v)1.2. PKA is bound to Ca(v)1.2 within a macromolecular signaling complex consisting of the beta(2) adrenergic receptor, trimeric G(s) protein, and adenylyl cyclase for fast, localized, and hence specific signaling [Davare, M. A., Avdonin, V., Hall, D. D., Peden, E. M., Buret, A., Weinberg, R. J., Horne, M. C., Hoshi, T., and Hell, J. W. (2001) Science 293, 98-101]. Protein phosphatase 2A (PP2A) serves to effectively balance serine 1928 phosphorylation by PKA through its association with the Ca(v)1.2 complex [Davare, M. A., Horne, M. C., and Hell, J. W. (2000) J. Biol. Chem. 275, 39710-39717]. We now show that native PP2A holoenzymes, as well as the catalytic subunit itself, bind to alpha(1)1.2 immediately downstream of serine 1928. Of those holoenzymes, only heterotrimeric PP2A containing B' and B' ' subunits copurify with alpha(1)1.2. Preventing the binding of PP2A by truncating alpha(1)1.2 28 residues downstream of serine 1928 hampers its dephosphorylation in intact cells. Our results demonstrate for the first time that a stable interaction of PP2A with Ca(v)1.2 is required for effective reversal of PKA-mediated channel phosphorylation. Accordingly, PKA as well as PP2A are constitutively associated with Ca(v)1.2 for its proper regulation by phosphorylation and dephosphorylation of serine 1928.     

Protein phosphatase 6 down-regulates TAK1 kinase activation in the IL-1 signaling pathway

TAK1 (transforming growth factor beta-activated kinase 1) is a serine/threonine kinase that is a mitogen-activated protein kinase kinase kinase and an essential intracellular signaling component in inflammatory signaling pathways. Upon stimulation of cells with inflammatory cytokines, TAK1 binds proteins that stimulate autophosphorylation within its activation loop and is thereby catalytically activated. This activation is transient; it peaks within a couple of minutes and is subsequently down-regulated rapidly to basal levels. The mechanism of down-regulation of TAK1 has not yet been elucidated. In this study, we found that toxin inhibition of type 2A protein phosphatases greatly enhances interleukin 1 (IL-1)-dependent phosphorylation of Thr-187 in the TAK1 activation loop as well as the catalytic activity of TAK1. From proteomic analysis of TAK1-binding proteins, we identified protein phosphatase 6 (PP6), a type-2A phosphatase, and demonstrated that PP6 associated with and inactivated TAK1 by dephosphorylation of Thr-187. Ectopic and endogenous PP6 co-precipitated with TAK1, and expression of PP6 reduced IL-1 activation of TAK1 but did not affect osmotic activation of MLK3, another MAPKKK. Reduction of PP6 expression by small interfering RNA enhances IL-1-induced phosphorylation of Thr-187 in TAK1. Enhancement occurred without change in levels of PP2A showing specificity for PP6. Our results demonstrate that PP6 specifically down-regulates TAK1 through dephosphorylation of Thr-187 in the activation loop, which is likely important for suppressing inflammatory responses via TAK1 signaling pathways.     

Association of the type 1 protein phosphatase PP1 with the A-kinase anchoring protein AKAP220

The cyclic AMP (cAMP)-dependent protein kinase (PKA) and the type 1 protein phosphatase (PP1) are broad-specificity signaling enzymes with opposing actions that catalyze changes in the phosphorylation state of cellular proteins. Subcellular targeting to the vicinity of preferred substrates is a means of restricting the specificity of each enzyme [1] [2]. Compartmentalization of the PKA holoenzyme is mediated through association of the regulatory subunits with A-kinase anchoring proteins (AKAPs), whereas a diverse family of phosphatase-targeting subunits directs the location of the PP1 catalytic subunit (PP1c) [3] [4]. Here, we demonstrate that the PKA-anchoring protein, AKAP220, binds PP1c with a dissociation constant (KD) of 12.1 +/- 4 nM in vitro. Immunoprecipitation of PP1 from cell extracts resulted in a 10.4 +/- 3.8-fold enrichment of PKA activity. AKAP220 co-purified with PP1c by affinity chromatography on microcystin sepharos Immunocytochemical analysis demonstrated that the kinase, the phosphatase and the anchoring protein had distinct but overlapping staining patterns in rat hippocampal neurons. Collectively, these results provide the first evidence that AKAP220 is a multivalent anchoring protein that maintains a signaling scaffold of PP1 and the PKA holoenzyme.     

Role for protein phosphatase 2A in the regulation of Calu-3 epithelial Na+-K+-2Cl-, type 1 co-transport function

Activity of Na+-K+-2Cl- co-transport (NKCC1) in epithelia is thought to be highly regulated through phosphorylation and dephosphorylation of the transporter. Previous functional studies from this laboratory suggested a role for protein phosphatase 2A (PP2A) as a serine/threonine protein phosphatase involved in the regulation of mammalian tracheal epithelial NKCC1. We expand on these studies to characterize serine/threonine protein phosphatase(s) necessary for regulation of NKCC1 function and the interaction of the phosphatase(s) with proteins associated with NKCC1. NKCC1 activity was measured as bumetanide-sensitive 86Rb uptake or basolateral to apical 86Rb flux in primary cultures of human tracheal epithelial cells or in Calu-3 airway epithelial cells grown on Transwell filter inserts. Preincubation with 0.1 nm okadaic acid, a PP2A > phosphatase 1 (PP1) inhibitor, increased NKCC1 activity 3.5-fold in human tracheal epithelial cells and 4.1-fold in Calu-3 cells. Calyculin, a PP1 > PP2A inhibitor, did not alter NKCC1 activity or percent bumetanide-sensitive flux. The effect of OA was dose-dependent with an IC50 of 0.4 nm. The alpha1-adrenergic agonist methoxamine increased NKCC1 activity and transiently increased PP2A activity 3.8-fold but did not alter PP1 activity. OA augmented methoxamine-dependent stimulation of NKCC1 activity. PP1, PP2A, and PP2C but not PP2B were detected in lysates from Calu-3 cells by immunoblot analysis. PP1 was not detected in immunoprecipitates of NKCC1 and vice versa. PP2A co-immunoprecipitated with NKCC1 and protein kinase C-delta (PKC-delta) and was pulled down by a recombinant N terminus of NKCC1 consisting of amino acids 1-286. One novel finding is co-precipitation of STE20-related proline-alanine-rich kinase, a regulatory kinase for NKCC1, with PP2A and PKC-delta. The results suggest a model of actin serving as a scaffold for binding and association of PKC-delta, PP2A, and STE20-related proline-alanine-rich kinase. The role of the complex of serine/threonine protein kinases and a protein phosphatase is probably the maintenance of optimal phosphorylation of NKCC1 coincident with its physiological function in epithelial absorption and secretion.