Biomedical subjects

Anthony R Means

Publications and source records attributed to Anthony R Means.

At least 19 recordsLinked to original sources

PubMed2024

Convergence on CaMK4: A Key Modulator of Autism-Associated Signaling Pathways in Neurons.

Although the precise underlying cause(s) of autism spectrum disorder remain unclear, more than 1000 rare genetic variations are associated with the condition. For many people living with profound autism, this genetic heterogeneity has impeded the identification of common biological targets for therapy development for core and comorbid traits that include significant impairments in social communication and repetitive and restricted behaviors. A substantial number of genes associated with autism encode proteins involved in signal transduction and synaptic transmission that are critical for brain development and function. CAMK4 is an emerging risk gene for autism spectrum disorder that encodes the CaMK4 (calcium/calmodulin-dependent protein kinase 4) enzyme. CaMK4 is a key component of a Ca2+-activated signaling pathway that regulates neurodevelopment and synaptic plasticity. In this review, we discuss 3 genetic variants of CAMK4 found in individuals with hyperkinetic movement disorder and comorbid neurological symptoms including autism spectrum disorder that are likely pathogenic with monogenic effect. We also comment on 4 other genetic variations in CAMK4 that show associations with autism spectrum disorder, as well as 12 examples of autism-associated variations in other genes that impact CaMK4 signaling pathways. Finally, we highlight 3 environmental risk factors that impact CaMK4 signaling based on studies of preclinical models of autism and/or clinical cohorts. Overall, we review molecular, genetic, physiological, and environmental evidence that suggest that defects in the CaMK4 signaling pathway may play an important role in a common autism pathogenesis network across numerous patient groups, and we propose CaMK4 as a potential therapeutic target.

Humans↗

PubMed2006

Regulation of AMP-activated protein kinase by multisite phosphorylation in response to agents that elevate cellular cAMP.

The AMP-activated protein kinase (AMPK) and cAMP signaling systems are both key regulators of cellular metabolism. In this study, we show that AMPK activity is attenuated in response to cAMP-elevating agents through modulation of at least two of its alpha subunit phosphorylation sites, viz. alpha-Thr(172) and alpha1-Ser(485)/alpha2-Ser(491), in the clonal beta-cell line INS-1 as well as in mouse embryonic fibroblasts and COS cells. Forskolin, isobutylmethylxanthine, and the glucose-dependent insulinotropic peptide inhibited AMPK activity and reduced phosphorylation of the activation loop alpha-Thr(172) via inhibition of calcium/calmodulin-dependent protein kinase kinase-alpha and -beta, but not LKB1. These agents also enhanced phosphorylation of alpha-Ser(485/491) by the cAMP-dependent protein kinase. AMPK alpha-Ser(485/491) phosphorylation was necessary but not sufficient for inhibition of AMPK activity in response to forskolin/isobutylmethylxanthine. We show that AMPK alpha-Ser(485/491) can be a site for autophosphorylation, which may play a role in limiting AMPK activation in response to energy depletion or other regulators. Thus, our findings not only demonstrate cross-talk between the cAMP/cAMP-dependent protein kinase and AMPK signaling modules, but also describe a novel mechanism by which multisite phosphorylation of AMPK contributes to regulation of its enzyme activity.

1-Methyl-3-isobutylxanthine↗

PubMed2006

Rho kinase differentially regulates phosphorylation of nonmuscle myosin II isoforms A and B during cell rounding and migration.

The actin-myosin cytoskeleton is generally accepted to produce the contractile forces necessary for cellular processes such as cell rounding and migration. All vertebrates examined to date are known to express at least two isoforms of non-muscle myosin II, referred to as myosin IIA and myosin IIB. Studies of myosin IIA and IIB in cultured cells and null mice suggest that these isoforms perform distinct functions. However, how each myosin II isoform contributes individually to all the cellular functions attributed to "myosin II" has yet to be fully characterized. Using isoform-specific small-interfering RNAs, we found that depletion of either isoform resulted in opposing migration phenotypes, with myosin IIA- and IIB-depleted cells exhibiting higher and lower wound healing migration rates, respectively. In addition, myosin IIA-depleted cells demonstrated impaired thrombin-induced cell rounding and undertook a more motile morphology, exhibiting decreased amounts of stress fibers and focal adhesions, with concomitant increases in cellular protrusions. Cells depleted of myosin IIB, however, were efficient in thrombin-induced cell rounding, displayed a more retractile phenotype, and maintained focal adhesions but only in the periphery. Last, we present evidence that Rho kinase preferentially regulates phosphorylation of the regulatory light chain associated with myosin IIA. Our data suggest that the myosin IIA and IIB isoforms are regulated by different signaling pathways to perform distinct cellular activities and that myosin IIA is preferentially required for Rho-mediated contractile functions.

Cell Line, Tumor↗

PubMed2006

Ubiquitylation of cyclin E requires the sequential function of SCF complexes containing distinct hCdc4 isoforms.

Cyclin E, an activator of cyclin-dependent kinase 2 (Cdk2), is targeted for proteasomal degradation by phosphorylation-dependent multiubiquitylation via the ubiquitin ligase SCF(hCdc4). SCF ubiquitin ligases are composed of a core of conserved subunits and one variable subunit (an F box protein) involved in substrate recognition. We show here that multiubiquitylation of cyclin E requires the sequential function of two distinct splice variant isoforms of the F box protein hCdc4 known as alpha and gamma. SCF(hCdc4alpha) binds a complex containing cyclin E, Cdk2, and the prolyl cis/trans isomerase Pin1 and promotes the activity of Pin1 without directly ubiquitylating cyclin E. However, due to the action of this SCF(hCdc4alpha)-Pin1 complex, cyclin E becomes an efficient ubiquitylation substrate of SCF(hCdc4gamma). Furthermore, in the context of Cdc4alpha and cyclin E, mutational data suggest that Pin1 isomerizes a noncanonical proline-proline bond, with the possibility that Cdc4alpha may serve as a cofactor for altering the specificity of Pin1.

Binding Sites↗

PubMed2005

Chutes and Ladders: the search for protein kinases that act on AMPK.

AMP-activated protein kinase (AMPK), a key regulator of energy homeostasis in mammalian cells, is, in turn, regulated by long-sought upstream protein kinases (AMPKKs). Following the recent identification of the tumor-suppressor kinase LKB1 as an AMPKK, a broader role for AMPK in metabolic economy has been unveiled by a new body of work from three groups that implicates the Ca(2+)/calmodulin-dependent protein kinase kinases as AMPKKs. We suggest that PKE (protein kinase "energy" or "economy") is now an apt name for this kinase, which regulates both cellular and whole-organism energy homeostasis.

AMP-Activated Protein Kinase Kinases↗

PubMed2005

The loss of PIN1 deregulates cyclin E and sensitizes mouse embryo fibroblasts to genomic instability.

During the G0/G1-S phase transition, the timely synthesis and degradation of key regulatory proteins is required for normal cell cycle progression. Two of these proteins, c-Myc and cyclin E, are recognized by the Cdc4 E3 ligase of the Skp1/Cul1/Rbx1 (SCF) complex. SCF(Cdc4) binds to a similar phosphodegron sequence in c-Myc and cyclin E proteins resulting in ubiquitylation and degradation of both proteins via the 26 S proteosome. Since the prolyl isomerase Pin1 binds the c-Myc phosphodegron and participates in regulation of c-Myc turnover, we hypothesized that Pin1 would bind to and regulate cyclin E turnover in a similar manner. Here we show that Pin1 regulates the turnover of cyclin E in mouse embryo fibroblasts. Pin1 binds to the cyclin E-Cdk2 complex in a manner that depends on Ser384 of cyclin E, which is phosphorylated by Cdk2. The absence of Pin1 results in an increased steady-state level of cyclin E and stalling of the cells in the G1/S phase of the cell cycle. The cellular changes that result from the loss of Pin1 predispose Pin1 null mouse embryo fibroblasts to undergo more rapid genomic instability when immortalized by conditional inactivation of p53 and sensitizes these cells to more aggressive Ras-dependent transformation and tumorigenesis.

Animals↗

PubMed2005

Calmodulin-dependent protein kinase IV regulates hematopoietic stem cell maintenance.

The hematopoietic stem cell (HSC) gives rise to all mature, terminally differentiated cells of the blood. Here we show that calmodulin-dependent protein kinase IV (CaMKIV) is present in c-Kit+ ScaI+ Lin(-/low) hematopoietic progenitor cells (KLS cells) and that its absence results in hematopoietic failure, characterized by a diminished KLS cell population and by an inability of these cells to reconstitute blood cells upon serial transplantation. KLS cell failure in the absence of CaMKIV is correlated with increased apoptosis and proliferation of these cells in vivo and in vitro. In turn, these cell biological defects are correlated with decreases in CREB-serine 133 phosphorylation as well as in CREB-binding protein (CBP) and Bcl-2 levels. Re-expression of CaMKIV in Camk4-/- KLS cells results in the rescue of the proliferation defects in vitro as well as in the restoration of CBP and Bcl-2 to wild type levels. These studies show that CaMKIV is a regulator of HSC homeostasis and suggest that its effects may be in part mediated via regulation of CBP and Bcl-2.

Animals↗

PubMed2005

The Ca2+/calmodulin-dependent protein kinase kinases are AMP-activated protein kinase kinases.

The AMP-activated protein kinase (AMPK) is an important regulator of cellular metabolism in response to metabolic stress and to other regulatory signals. AMPK activity is absolutely dependent upon phosphorylation of AMPKalphaThr-172 in its activation loop by one or more AMPK kinases (AMPKKs). The tumor suppressor kinase, LKB1, is a major AMPKK present in a variety of tissues and cells, but several lines of evidence point to the existence of other AMPKKs. We have employed three cell lines deficient in LKB1 to study AMPK regulation and phosphorylation, HeLa, A549, and murine embryo fibroblasts derived from LKB(-/-) mice. In HeLa and A549 cells, mannitol, 2-deoxyglucose, and ionomycin, but not 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), treatment activates AMPK by alphaThr-172 phosphorylation. These responses, as well as the downstream effects of AMPK on the phosphorylation of acetyl-CoA carboxylase, are largely inhibited by the Ca(2+)/ calmodulin-dependent protein kinase kinase (CaMKK) inhibitor, STO-609. AMPKK activity in HeLa cell lysates measured in vitro is totally inhibited by STO-609 with an IC50 comparable with that of the known CaMKK isoforms, CaMKKalpha and CaMKKbeta. Furthermore, 2-deoxyglucose- and ionomycin-stimulated AMPK activity, alphaThr-172 phosphorylation, and acetyl-CoA carboxylase phosphorylation are substantially reduced in HeLa cells transfected with small interfering RNAs specific for CaMKKalpha and CaMKKbeta. Lastly, the activation of AMPK in response to ionomycin and 2-deoxyglucose is not impaired in LKB1(-/-) murine embryo fibroblasts. These data indicate that the CaMKKs function in intact cells as AMPKKs, predicting wider roles for these kinases in regulating AMPK activity in vivo.

AMP-Activated Protein Kinase Kinases↗

PubMed2005

Calmodulin-dependent protein kinase IV regulates nuclear export of Cabin1 during T-cell activation.

Calcium signaling is critical for activation of T lymphocytes and has been proposed to be transduced through multiple calmodulin target proteins. Whereas the calcineurin-NFAT signaling module is critical for all mammalian T cells, the role of calmodulin-dependent kinase IV (CaMKIV) in mouse naïve CD4+ T-cell activation remains enigmatic. We have applied lentivius-mediated RNA interference of CaMKIV to human T cells and found that knockdown of CaMKIV abrogates T-cell receptor-mediated transcription of the IL-2 gene. We demonstrate that CaMKIV directly phosphorylates Cabin1, a transcriptional corepressor for myocyte enhancer factor 2, creating a docking site for 14-3-3, which causes its nuclear export. CaMKIV-mediated nuclear export of Cabin1 is likely to account for a significant part of the requirement of CaMKIV during human T-cell activation.

14-3-3 Proteins↗

PubMed2005

The autonomous activity of calcium/calmodulin-dependent protein kinase IV is required for its role in transcription.

Calcium/calmodulin-dependent kinase IV (CaMKIV) is a multifunctional serine/threonine kinase that is positively regulated by two main events. The first is the binding of calcium/calmodulin (Ca(2+)/CaM), which relieves intramolecular autoinhibition of the enzyme and leads to basal kinase activity. The second is activation by the upstream kinase, Ca(2+)/calmodulin-dependent kinase kinase. Phosphorylation of Ca(2+)/CaM-bound CaMKIV on its activation loop threonine (residue Thr(200) in human CaMKIV) by Ca(2+)/calmodulin-dependent kinase kinase leads to increased CaMKIV kinase activity. It has also been repeatedly noted that activation of CaMKIV is accompanied by the generation of Ca(2+)/CaM-independent or autonomous activity, although the significance of this event has been unclear. Here we demonstrate the importance of autonomous activity to CaMKIV biological function. We show that phosphorylation of CaMKIV on Thr(200) leads to the generation of a fully Ca(2+)/CaM-independent enzyme. By analyzing the behavior of wild-type and mutant CaMKIV proteins in biochemical experiments and cellular transcriptional assays, we demonstrate that CaMKIV autonomous activity is necessary and sufficient for CaMKIV-mediated transcription. The ability of wild-type CaMKIV to drive cAMP response element-binding protein-mediated transcription is strictly dependent upon an initiating Ca(2+) stimulus, which leads to kinase activation and development of autonomous activity in cells. Mutant CaMKIV proteins that are incapable of developing autonomous activity within a cellular context fail to drive transcription, whereas certain CaMKIV mutants that possess constitutive autonomous activity drive transcription in the absence of a Ca(2+) stimulus and independent of Ca(2+)/CaM binding or Thr(200) phosphorylation.

Amino Acid Sequence↗

PubMed2005

Peptidyl-prolyl isomerase 1 (Pin1) serves as a coactivator of steroid receptor by regulating the activity of phosphorylated steroid receptor coactivator 3 (SRC-3/AIB1).

Steroid receptor coactivator 3 (SRC-3/AIB1) interacts with steroid receptors in a ligand-dependent manner to activate receptor-mediated transcription. A number of intracellular signaling pathways initiated by growth factors and hormones induce phosphorylation of SRC-3, regulating its function and contributing to its oncogenic potential. However, the range of mechanisms by which phosphorylation affects coactivator function remains largely undefined. We demonstrate here that peptidyl-prolyl isomerase 1 (Pin1), which catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds to induce conformational changes of its target proteins, interacts selectively with phosphorylated SRC-3. In addition, Pin1 and SRC-3 activate nuclear-receptor-regulated transcription synergistically. Depletion of Pin1 by small interfering RNA (siRNA) reduces hormone-dependent transcription from both transfected reporters and an endogenous steroid receptor target gene. We present evidence that Pin1 modulates interactions between SRC-3 and CBP/p300. The interaction is enhanced in vitro and in vivo by Pin1 and diminished when cellular Pin1 is reduced by siRNA or in stable Pin1-depleted cell lines. Depletion of Pin1 in MCF-7 human breast cancer cells reduces the endogenous estrogen-dependent recruitment of p300 to the promoters of estrogen receptor-dependent genes. Pin1 overexpression enhanced SRC-3 cellular turnover, and depletion of Pin1 stabilized SRC-3. Our results suggest that Pin1 functions as a transcriptional coactivator of nuclear receptors by modulating SRC-3 coactivator protein-protein complex formation and ultimately by also promoting the turnover of the activated SRC-3 oncoprotein.

Acetyltransferases↗

PubMed2004

A role for Pin1 in mammalian germ cell development and spermatogenesis.

The peptidyl-prolyl isomerase Pin1 is proposed to have diverse functions in many vital aspects of the cell. Despite the multitude of proteins targeted by Pin1 and the proposed regulatory role it plays in critical cellular functions, Pin1 is an essential gene in some eukaryotic organisms, but is dispensable in metazoans. In two genetic models, Candida albicans and Drosophila melanogaster, Pin1 participates in distinct developmental processes regulated by the MAPK pathway. Pin1-deficient mice exhibit decreased primordial germ cell proliferation during embryonic development, along with several degenerative or proliferative defects in the adult testis, retina, mammary gland, and brain. The combination of primordial germ cell deficit and spermatogonial depletion contributes to severe fertility defects in Pin1-null mice. Since growth factor activated MAPK pathways are vital to germ cell proliferation and differentiation, a role for Pin1 in mammalian germ cell development and spermatogenesis is discussed in the context of the Ras/MEK/MAPK pathway.

Animals↗

PubMed2004

PINA is essential for growth and positively influences NIMA function in Aspergillus nidulans.

The phospho-Ser/Thr-directed prolyl-isomerase Pin1 was originally identified in vertebrate systems as a negative regulator of NIMA, a Ser/Thr protein kinase that regulates the G(2)/M transition in Aspergillus nidulans. Here we explore the physiological roles of the Pin1 orthologue, PINA, in A. nidulans and evaluate the relevance of the interaction of PINA with NIMA in this fungus. We find pinA to be an essential gene in A. nidulans. In addition, when PINA levels are reduced 50-fold the cells grow at a reduced rate. Upon germination under conditions that repress PINA expression, the cells are delayed in the interphase activation of NIMX(cdc2), whereas they traverse the other phases of the cell cycle at a similar rate to controls. These results indicate that a marked reduction of PINA results in a lengthening of G(1). Additionally, PINA repression increases the rate at which the cells enter mitosis following release from a hydroxyurea arrest without altering the sensitivity of the fungus to agents that activate the replication or DNA damage checkpoints. In contrast to predictions based on Pin1, the physical interaction between PINA and NIMA is primarily dependent upon the prolylisomerase domain of PINA and the C-terminal 303 amino acids of NIMA. Finally, reduction of PINA levels exacerbates the nimA5 temperature-sensitive mutant, whereas overexpression of PINA decreases the severity of this mutation, results that are consistent with a positive genetic interaction between PINA and NIMA. Thus, although PINA is essential and positively regulates NIMA function, A. nidulans is most sensitive to a reduction in PINA concentration in G(1) rather than in G(2)/M.

Adenosine Triphosphatases↗

PubMed2004

Regulation and function of the calcium/calmodulin-dependent protein kinase IV/protein serine/threonine phosphatase 2A signaling complex.

Calcium/calmodulin-dependent protein kinase IV (CaMKIV) is a member of the broad substrate specificity class of Ca(2+)/calmodulin (CaM)-dependent protein kinases and functions as a potent stimulator of Ca(2+)-dependent gene expression. Activation of CaMKIV is a transient, tightly regulated event requiring both Ca(2+)/CaM binding and phosphorylation of the kinase on T200 by an upstream CaMK kinase (CaMKK). Previously, CaMKIV was shown to stably associate with protein serine/threonine phosphatase 2A (PP2A), which was proposed to play a role in negatively regulating the kinase. Here we report that the Ca(2+)/CaM binding-autoinhibitory domain of CaMKIV is required for association of the kinase with PP2A and that binding of PP2A and Ca(2+)/CaM appears to be mutually exclusive. We demonstrate that inhibition of the CaMKIV/PP2A association in cells results in enhanced CaMKIV-mediated gene transcription that is independent of Ca(2+)/CaM. The enhanced transcriptional activity correlates with the elevated level of phospho-T200 that accumulates when CaMKIV is prevented from interacting with PP2A. Collectively, these data suggest a molecular basis for the sequential activation and inactivation of CaMKIV. First, in response to an increase in intracellular Ca(2+), CaMKIV binds Ca(2+)/CaM and becomes phosphorylated on T200 by CaMKK. These events result in the generation of autonomous activity required for CaMKIV-mediated transcriptional regulation. The CaMKIV-associated PP2A then dephosphorylates CaMKIV T200, thereby terminating autonomous activity and CaMKIV-mediated gene transcription.

Amino Acid Sequence↗

PubMed2004

A signalling pathway controlling c-Myc degradation that impacts oncogenic transformation of human cells.

The stability of c-Myc is regulated by multiple Ras effector pathways. Phosphorylation at Ser 62 stabilizes c-Myc, whereas subsequent phosphorylation at Thr 58 is required for its degradation. Here we show that Ser 62 is dephosphorylated by protein phosphatase 2A (PP2A) before ubiquitination of c-Myc, and that PP2A activity is regulated by the Pin1 prolyl isomerase. Furthermore, the absence of Pin1 or inhibition of PP2A stabilizes c-Myc. A stable c-Myc(T58A) mutant that cannot bind Pin1 or be dephosphorylated by PP2A replaces SV40 small T antigen in human cell transformation and tumorigenesis assays. Therefore, small T antigen, which inactivates PP2A, exerts its oncogenic potential by preventing dephosphorylation of c-Myc, resulting in c-Myc stabilization. Thus, Ras-dependent signalling cascades ensure transient and self-limiting accumulation of c-Myc, disruption of which contributes to human cell oncogenesis.

Amino Acid Sequence↗

PubMed2004

Calcineurin regulates cyclin D1 accumulation in growth-stimulated fibroblasts.

Calcium (Ca(2+)) and calmodulin (CaM) are required for progression of mammalian cells from quiescence into S phase. In multiple cell types, cyclosporin A causes a G(1) cell cycle arrest, implicating the serine/threonine phosphatase calcineurin as one Ca(2+)/CaM-dependent enzyme required for G(1) transit. Here, we show, in diploid human fibroblasts, that cyclosporin A arrested cells in G(1) before cyclin D/cdk4 complex activation and retinoblastoma hyperphosphorylation. This arrest occurred in early G(1) with low levels of cyclin D1 protein. Because cyclin D1 mRNA was induced normally in the cyclosporin A-treated cells, we analyzed the half-life of cyclin D1 in the presence of cyclosporin A and found no difference from control cells. However, cyclosporin A treatment dramatically reduced cyclin D1 protein synthesis. Although these pharmacological experiments suggested that calcineurin regulates cyclin D1 synthesis, we evaluated the effects of overexpression of activated calcineurin on cyclin D1 synthesis. In contrast to the reduction of cyclin D1 with cyclosporin A, ectopic expression of calcium/calmodulin-independent calcineurin promoted synthesis of cyclin D1 during G(1) progression. Therefore, calcineurin is a Ca(2+)/CaM-dependent target that regulates cyclin D1 accumulation in G(1).

Blotting, Northern↗

PubMed2004

Regulation of cyclin D1/Cdk4 complexes by calcium/calmodulin-dependent protein kinase I.

The selective inhibitor of the multifunctional calcium/calmodulin-dependent kinases (CaMK), KN-93, arrests a variety of cell types in G(1). However, the biochemical nature of this G(1) arrest point and the physiological target of KN-93 in G(1) remain controversial. Here we show that in WI-38 human diploid fibroblasts KN-93 reversibly arrested cells in late G(1) prior to detectable cyclin-dependent kinase 4 (cdk4) activation. At the KN-93 arrest point, we found that cyclin D1/cdk4 complexes had assembled with p21/p27, accumulated in the nucleus, and become phosphorylated on Thr-172, yet were relatively inactive. Additional examination of cdk4 complexes by gel filtration analysis demonstrated that, in late G(1), cyclin D1-containing complexes migrated toward lower molecular weight (M(r)) fractions and this altered migration was accompanied by the appearance of two peaks of cdk4 activity, at 150-200 and 70 kDa, respectively. KN-93 prevented both the activation of cdk4, and this shift in cyclin D1 migration and overexpression of cyclin D1/cdk4 overcame the KN-93 arrest. To determine which multifunctional CaMK acts in G(1), we expressed kinase-deficient forms of CaMKI and CaMKII. Overexpression of kinase-deficient CaMKI, but not CaMKII, prevented cdk4 activation, mimicking the KN-93 arrest point. Therefore, we hypothesize that KN-93 prevents a very late, uncharacterized step in cyclin D/cdk4 activation that involves CaMKI and follows complex assembly, nuclear entry, and phosphorylation.

Adenoviridae↗

PubMed2003

Catalytic activity is required for calcium/calmodulin-dependent protein kinase IV to enter the nucleus.

Calcium/calmodulin-dependent protein kinase IV (CaMKIV) is a nuclear protein kinase that responds to acute rises in intracellular calcium by phosphorylating and activating proteins involved in transcription. Consistent with these roles, CaMKIV is found predominantly in the nucleus of cells in which it is expressed. Here we evaluate nuclear entry of CaMKIV and demonstrate that the protein kinase homology domain is both necessary and sufficient for nuclear localization. Unexpectedly, although catalytic activity is required for nuclear translocation, it is not required for CaMKIV to interact with the nuclear adaptor protein, importin-alpha. Because the catalytically inactive molecules remain in the cytoplasm, these data suggest that this interaction is not sufficient for nuclear entry. We evaluated a role for other proteins known to interact with CaMKIV in regulation of its nuclear entry. Although our data do not support a role for calmodulin or protein phosphatase 2A, the catalytically inactive CaMKIV proteins interact more avidly with CaM-dependent protein kinase kinase (CaMKK), which is restricted to the cytoplasm. We find that the catalytically inactive proteins do not inhibit nuclear entry of wild-type CaMKIV but do inhibit the ability of the wild-type protein kinase to stimulate cyclic AMP response element-binding protein-mediated transcription. Because activation loop phosphorylation is required for the transcriptional roles of CaMKIV, these data suggest that CaMKK phosphorylation of CaMKIV may occur in the cytoplasm. We propose that sequestration of CaMKK may be the molecular mechanism by which catalytically inactive mutants of CaMKIV exert their "dominant-negative" functions within the cell.

Calcium-Calmodulin-Dependent Protein Kinase Type 4↗