Martha A. Bass

Death, Cardiac Dysfunction, and Arrhythmias Are Increased by Calmodulin Kinase II in Calcineurin Cardiomyopathy

Background— Activation of cellular Ca2+ signaling molecules appears to be a fundamental step in the progression of cardiomyopathy and arrhythmias. Myocardial overexpression of the constitutively active Ca2+-dependent phosphatase calcineurin (CAN) causes severe cardiomyopathy marked by left ventricular (LV) dysfunction, arrhythmias, and increased mortality rate, but CAN antagonist drugs primarily reduce hypertrophy without improving LV function or risk of death. Methods and Results— We found that activity and expression of a second Ca2+-activated signaling molecule, calmodulin kinase II (CaMKII), were increased in hearts from CAN transgenic mice and that CaMKII-inhibitory drugs improved LV function and suppressed arrhythmias. We devised a genetic approach to “clamp” CaMKII activity in CAN mice to control levels by interbreeding CAN transgenic mice with mice expressing a specific CaMKII inhibitor in cardiomyocytes. We developed transgenic control mice by interbreeding CAN transgenic mice with mice expressing an inactive version of the CaMKII-inhibitory peptide. CAN mice with CaMKII inhibition had reduced risk of death and increased LV and ventricular myocyte function and were less susceptible to arrhythmias. CaMKII inhibition did not reduce transgenic overexpression of CAN or expression of endogenous CaMKII protein or significantly reduce most measures of cardiac hypertrophy. Conclusions— CaMKII is a downstream signal in CAN cardiomyopathy, and increased CaMKII activity contributes to cardiac dysfunction, arrhythmia susceptibility, and longevity during CAN overexpression.

Brain Actin-associated Protein Phosphatase 1 Holoenzymes Containing Spinophilin, Neurabin, and Selected Catalytic Subunit Isoforms

We previously characterized PP1bp134 and PP1bp175, two neuronal proteins that bind the protein phosphatase 1 catalytic subunit (PP1). Here we purify from rat brain actin-cytoskeletal extracts PP1A holoenzymes selectively enriched in PP1γ1 over PP1β isoforms and also containing PP1bp134 and PP1bp175. PP1bp134 and PP1bp175 were identified as the synapse-localized F-actin-binding proteins spinophilin (Allen, P. B., Ouimet, C. C., and Greengard, P. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9956–9561; Satoh, A., Nakanishi, H., Obaishi, H., Wada, M., Takahashi, K., Satoh, K., Hirao, K., Nishioka, H., Hata, Y., Mizoguchi, A., and Takai, Y. (1998) J. Biol. Chem. 273, 3470–3475) and neurabin (Nakanishi, H., Obaishi, H., Satoh, A., Wada, M., Mandai, K., Satoh, K., Nishioka, H., Matsuura, Y., Mizoguchi, A., and Takai, Y. (1997)J. Cell Biol. 139, 951–961), respectively. Recombinant spinophilin and neurabin interacted with endogenous PP1 and also with each other when co-expressed in HEK293 cells. Spinophilin residues 427–470, or homologous neurabin residues 436–479, were sufficient to bind PP1 in gel overlay assays, and selectively bound PP1γ1 from a mixture of brain protein phosphatase catalytic subunits; additional N- and C-terminal sequences were required for potent inhibition of PP1. Immunoprecipitation of spinophilin or neurabin from crude brain extracts selectively coprecipitated PP1γ1 over PP1β. Moreover, immunoprecipitation of PP1γ1 from brain extracts efficiently coprecipitated spinophilin and neurabin, whereas PP1β immunoprecipitation did not. Thus, PP1A holoenzymes containing spinophilin and/or neurabin target specific neuronal PP1 isoforms, facilitating efficient regulation of synaptic phosphoproteins.

Association of calcium/calmodulin-dependent kinase II with developmentally regulated splice variants of the postsynaptic density protein densin-180 *

In a continuing search for proteins that target calcium/calmodulin-dependent protein kinase II (CaMKII) to postsynaptic density (PSD) substrates important in synaptic plasticity, we showed that the PSD protein densin-180 binds CaMKII. Four putative splice variants (A–D) of the cytosolic tail of densin-180 are shown to be differentially expressed during brain development. Densin-180 splicing affects CaMKII phosphorylation of specific serine residues. Variants A, B, and D, but not C, bind CaMKII stoichiometrically and with high affinity, mediated by a differentially spliced domain. Densin-180 differs from the previously identified CaMKII-binding protein NR2B in that binding does not strictly require CaMKII autophosphorylation. Binding of densin-180 and NR2B to CaMKII is noncompetitive, indicating different interaction sites on CaMKII. Expression of the membrane-targeted CaMKII-binding domain of densin-180 confers membrane localization to coexpressed CaMKII without requiring calcium mobilization, suggesting that densin-180 plays a role in the constitutive association of CaMKII with PSDs.

Differential modulation of Ca2+/calmodulin-dependent protein kinase II activity by regulated interactions with N-methyl-D-aspartate receptor NR2B subunits and α-actinin

Neuronal Ca2+/calmodulin-dependent protein kinase II (CaMKII) interacts with several prominent dendritic spine proteins, which have been termed CaMKII-associated proteins. The NR2B subunit of N-methyl-d-aspartate (NMDA)-type glutamate receptor, densin-180, and α-actinin bind comparable, approximately stoichiometric amounts of Thr286-autophosphorylated CaMKIIα, forming a ternary complex (Robison, A. J., Bass, M. A., Jiao, Y., Macmillan, L. B., Carmody, L. C., Bartlett, R. K., and Colbran, R. J. (2005) J. Biol. Chem. 280, 35329-35336), but their impacts on CaMKII function are poorly understood. Here we show that these interactions are differentially regulated and exert distinct effects on CaMKII activity. Nonphosphorylated and Thr286-autophosphorylated CaMKII bind to α-actinin with similar efficacy, but autophosphorylation at Thr305/306 or Ca2+/calmodulin binding significantly reduce this binding. Moreover, α-actinin antagonizes CaMKII activation by Ca2+/calmodulin, as assessed by autophosphorylation and phosphorylation of a peptide substrate. CaMKII binding to densin (1247-1542) is partially independent of Thr286 autophosphorylation and is unaffected by Ca2+-independent autophosphorylation or Ca2+/calmodulin. In addition, the CaMKII binding domain of densin-180 has little effect on CaMKII activity. In contrast, the interaction of CaMKIIα with NR2B requires either Thr286 autophosphorylation or the binding of both Ca2+/calmodulin and adenine nucleotides. NR2B inhibits both the Ca2+/calmodulin-dependent and autonomous activities of CaMKII by a mechanism that is competitive with autocamtide-2 substrate, non-competitive with syntide-2 substrate, and uncompetitive with respect to ATP. In combination, these data suggest that dynamically regulated interactions with CaMKII-associated proteins could play pleiotropic roles in finetuning CaMKII signaling in defined subcellular compartments.

Association of brain protein phosphatase 1 with cytoskeletal targeting/regulatory subunits.

Abstract: Protein phosphatase 1 catalytic subunit (PP1C) is highly enriched in isolated rat postsynaptic densities. Gel overlay analyses using digoxigenin (DIG)‐labeled PP1C revealed four major rat brain PP1C‐binding proteins (PP1bps) with molecular masses of ≈216, 175, 134, and 75 kDa, which were (1) more abundant in brain than other rat tissues; (2) differentially expressed in microdissected brain regions; and (3) enriched in isolated cortex postsynaptic densities. PP1bp175, PP1bp134, PP1bp75, and PP1C were partially released from forebrain particulate extracts by incubation at low ionic strength, which destabilizes the actin cytoskeleton. Size‐exclusion chromatography of solubilized extracts separated two main PP1 activities (≈600 and ≈100 kDa). PP1bps and PP1Cγ1 were enriched in the ≈600‐kDa peak, but PP1Cβ was enriched in the ≈100‐kDa peak. Furthermore, PP1bp175 and PP1bp134 exhibited lower binding of recombinant DIG‐PP1Cβ than recombinant DIG‐PP1Cγ1 or DIG‐PP1Cα. Solubilized PP1bp175 and PP1bp134 interact with PP1C under native conditions, because they both (1) coeluted from size‐exclusion and ion‐exchange columns; (2) bound to microcystin‐LR‐Sepharose; and (3) coprecipitated using PP1C antibodies. Trypsinolysis of the ≈600‐kDa form of PP1 increased phosphorylase a phosphatase activity approximately fourfold, suggesting that interaction of PP1C with these PP1bps modulates its activity. Thus, brain PP1 activity is likely targeted to the cytoskeleton, including postsynaptic densities, by isoform‐selective binding of PP1C to these targeting/regulatory subunits, contributing to the specificity of its physiological roles.

A Protein Phosphatase-1γ1 Isoform Selectivity Determinant in Dendritic Spine-associated Neurabin

Protein phosphatase-1 (PP1) catalytic subunit isoforms interact with diverse proteins, typically containing a canonical (R/K)(V/I)XF motif. Despite sharing ∼90% amino acid sequence identity, PP1β and PP1γ1 have distinct subcellular localizations that may be determined by selective interactions with PP1-binding proteins. Immunoprecipitation studies from brain and muscle extracts demonstrated that PP1γ1 selectively interacts with spinophilin and neurabin, F-actin-targeting proteins, whereas PP1β selectively interacted with GM/RGL, the striated-muscle glycogen-targeting subunit. Glutathione S-transferase (GST) fusion proteins containing residues 146–493 of neurabin (GST-Nb-(146–493)) or residues 1–240 of GM/RGL (GST-GM-(1–240)) recapitulated these isoform selectivities in binding and phosphatase activity inhibition assays. Site-directed mutagenesis indicated that this isoform selectivity was not due to sequence differences between the canonical PP1-binding motifs (neurabin, 457KIKF460; GM/RGL, 65RVSF68). A chimeric GST fusion protein containing residues 1–64 of GM/RGL fused to residues 457–493 of neurabin (GST-GM/Nb) selectively bound to and inhibited PP1γ1, whereas a GST-Nb/GM chimera containing Nb-(146–460) fused to GM-(69–240) selectively interacted with and weakly inhibited PP1β, implicating domain(s) C-terminal to the (R/K)(V/I)XF motif as determinants of PP1 isoform selectivity. Deletion of Pro464 and Ile465 in neurabin (ΔPI) to equally space a conserved cluster of amino acids from the (R/K)(V/I)XF motif as in GM/RGL severely compromised the ability of neurabin to bind and inhibit both isoforms but did not affect PP1γ1 selectivity. Further analysis of a series of C-terminal truncated GST-Nb-(146–493) proteins identified residues 473–479 of neurabin as containing a crucial PP1γ1-selectivity determinant. In combination, these data identify a novel PP1γ1-selective interaction domain in neurabin that may allow for selective regulation and/or subcellular targeting of PP1 isoforms.

Characterization of a Central Ca2+/Calmodulin-dependent Protein Kinase IIα/β Binding Domain in Densin That Selectively Modulates Glutamate Receptor Subunit Phosphorylation

The densin C-terminal domain can target Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) in cells. Although the C-terminal domain selectively binds CaMKIIα in vitro, full-length densin associates with CaMKIIα or CaMKIIβ in brain extracts and in transfected HEK293 cells. This interaction requires a second central CaMKII binding site, the densin-IN domain, and an “open” activated CaMKII conformation caused by Ca2+/calmodulin binding, autophosphorylation at Thr-286/287, or mutation of Thr-286/287 to Asp. Mutations in the densin-IN domain (L815E) or in the CaMKIIα/β catalytic domain (I205/206K) disrupt the interaction. The amino acid sequence of the densin-IN domain is similar to the CaMKII inhibitor protein, CaMKIIN, and a CaMKIIN peptide competitively blocks CaMKII binding to densin. CaMKII is inhibited by both CaMKIIN and the densin-IN domain, but the inhibition by densin is substrate-selective. Phosphorylation of a model peptide substrate, syntide-2, or of Ser-831 in AMPA receptor GluA1 subunits is fully inhibited by densin. However, CaMKII phosphorylation of Ser-1303 in NMDA receptor GluN2B subunits is not effectively inhibited by densin in vitro or in intact cells. Thus, densin can target multiple CaMKII isoforms to differentially modulate phosphorylation of physiologically relevant downstream targets.

Association of Protein Phosphatase 1γ1 with Spinophilin Suppresses Phosphatase Activity in a Parkinson Disease Model

Sustained nigrostriatal dopamine depletion increases the serine/threonine phosphorylation of multiple striatal proteins that play a role in corticostriatal synaptic plasticity, including Thr286 phosphorylation of calcium/calmodulin-dependent protein kinase IIα (CaMKIIα). Mechanisms underlying these changes are unclear, but protein phosphatases play a critical role in the acute modulation of striatal protein phosphorylation. Here we show that dopamine depletion for periods ranging from 3 weeks to 10 months significantly reduces the total activity of protein phosphatase (PP) 1, but not of PP2A, in whole lysates of rat striatum, as measured using multiple substrates, including Thr286-autophosphorylated CaMKIIα. Striatal PP1 activity is partially inhibited by a fragment of the PP1-binding protein neurabin-I, Nb-(146–493), because of the selective inhibition of the PP1γ1 isoform. The fraction of PP1 activity that is insensitive to Nb-(146–493) was unaffected by dopamine depletion, demonstrating that dopamine depletion specifically reduces the activity of PP1 isoforms that are sensitive to Nb-(146–493) (i.e. PP1γ1). However, total striatal levels of PP1γ1 or any other PP1 isoform were unaffected by dopamine depletion, and our previous studies showed that total levels of the PP1 regulatory/targeting proteins DARPP-32, spinophilin, and neurabin were also unchanged. Rather, co-immunoprecipitation experiments demonstrated that dopamine depletion increases the association of PP1γ1 with spinophilin in striatal extracts. In combination, these data demonstrate that striatal dopamine depletion inhibits a specific synaptic phosphatase by increasing PP1γ1 interaction with spinophilin, perhaps contributing to hyperphosphorylation of synaptic proteins and disruptions of synaptic plasticity and/or dendritic morphology.

Analysis of specific interactions of native protein phosphatase 1 isoforms with targeting subunits.

Expression of recombinant PP1 isoforms with fully authentic properties has proven to be a challenge for several laboratories. In order to circumvent this technical limitation in the investigation of isoform-specific roles for PP1, methods have been developed to analyze specific properties of native PP1 isoforms. The well-documented method of ethanol precipitation of tissue extracts has been used to dissociate phosphatase catalytic subunits from their endogenous regulatory subunits and other cellular proteins. Although very low levels of PP1 and PP2A regulatory subunits are sometimes detected in PPC preparations, they are not associated with their respective catalytic subunits because they do not copurify with the catalytic subunits on microcystin-Sepharose (Bauman & Colbran, not shown). Thus, the PPC preparation represents a mixture of native monomeric phosphatase catalytic subunits (including PP1 isoforms, PP2AC, PP4C, and PP6C) that can be used to analyze their interactions with other proteins. The methods described in this report rely on the availability of highly specific antibodies to PP1 isoforms. The sheep antibodies have previously proven effective for immunoblotting and immunoprecipitation, whereas rabbit antibodies have also been used for immunocytochemistry. This paper documents the use of these antibodies in Far-Western overlay and glutathione-agarose cosedimentation assays to investigate interactions of specific PP1 isoforms with recombinant fragments of PP1-targeting subunits (spinophilin, neurabin and GM). Moreover, covalent coupling of affinity-purified sheep antibodies to agarose provided a means for the immuno-isolation of PP1 beta and PP1 gamma 1 from the PPC preparation. Active catalytic subunits are recovered from the affinity resin using chaotropic agents, permitting for the first time the assessment of the effects of specific targeting subunits on activities of individual native PP1 isoforms. These methods have been used successfully to demonstrate that some PP1-interacting proteins discriminate among the isoforms. The isoform inhibition assays provide a measure of the binding equilibrium in the milieu of the phosphatase assay. For example, while some PP1-binding proteins inhibit native PP1 beta and native PP1 gamma 1 with equivalent potency (e.g., PKA-phosphorylated inhibitor-1), spinophilin, neurabin and GM differentiate between these two isoforms; spinophilin and neurabin fragments inhibit native PP1 gamma 1 approximately 20-fold more potently than they inhibit native PP1 beta (Fig. 4), whereas GM inhibits native PP1 beta more potently than native PP1 gamma 1 (not shown). Moreover, the activity of native PP1 gamma 1 is approximately 100-fold more sensitive to neurabin and spinophilin than is the activity of bacterially-expressed recombinant PP1 gamma 1 (Fig. 4). The interpretation of these inhibition assays is consistent with data obtained in Far-Western overlay (Fig. 2) and glutathione-agarose cosedimentation assays (Fig. 3), which assess more stable interactions of PP1 isoforms. Thus, spinophilin and neurabin selectively bind PP1 gamma 1 over PP1 beta, whereas GM is highly selective for PP1 beta. These data are consistent with previous experiments that showed spinophilin and neurabin are present in PP1 gamma 1 complexes in brain extracts, but not in PP1 beta complexes. Moreover, only PP1 beta has been identified in complexes with GM in muscle extracts, although these data did not exclude the possibility that other isoforms were also present. Presumably, these isoform-selective interactions confer different functions on PP1. In summary, we have developed methods that should prove useful in defining the isoform-selectivity of other PP1-targeting subunits. Moreover, these methods may be employed to identify domains in PP1-interacting proteins that confer isoform specificity. Similar strategies may also be used to explore interactions of protein phosphatase catalytic subunits with other proteins.

Selective targeting of the γ1 isoform of protein phosphatase 1 to F-actin in intact cells requires multiple domains in spinophilin and neurabin

Protein phosphatase 1 (PP1) catalytic subunits dephosphorylate specific substrates in discrete subcellular compartments to modulate many cellular processes. Canonical PPl‐binding motifs (R/K‐V/ I‐X‐F) in a family of proteins mediate subcellular targeting, and the amino acids that form the binding pocket for the canonical motif are identical in all PP1 isoforms. However, PPlγ1 but not PP1β is selectively localized to F‐actin‐rich dendritic spines in neurons. Although the F‐actin‐binding proteins neurabin I and spinophilin (neurabin II) also bind PP1, their role in PP1 isoform selective targeting in intact cells is poorly understood. We show here that spinophilin selectively targets PP1γ1, but not PP1β, to F‐actin‐rich cortical regions of intact cells. Mutation of a PP1γ1 selectivity determinant (N464EDYDRR 470 in spinophilin: conserved as residues 473–479 in neurabin) to VKDYDTW severely attenuated PP1γ1 interactions with neurabins in vitro and in cells and disrupted PP1γ1 targeting to F‐actin. This domain is not involved in the weaker interactions of neurabins with PP1γ. In contrast, mutation of the canonical PP1‐binding motif attenuated interactions of neurabins with both isoforms. Thus, selective targeting of PP1γ1 to F‐actin by neurabins in intact cells requires both the canonical PP1‐binding motif and an auxiliary PP1γ1‐selectivity determinant.— Carmody L. C., IIBaucum A. J., Bass, M. A., Colbran R.J. Selective targeting of the γ1 isoform of protein phosphatase 1 to F‐actin in intact cells requires multiple domains in spinophilin and neurabin. FASEB J. 22, 1660–1671 (2008)