Calmodulin-dependent kinase II mediates vascular smooth muscle cell proliferation and is potentiated by extracellular signal regulated kinase.

Vascular smooth muscle cell (VSMC) proliferation contributes to vascular remodeling in atherosclerosis and hypertension. Calcium-dependent signaling through calcium/calmodulin-dependent kinase II (CaMKII) and ERK1/2 activation plays an important role in the regulation of VSMC proliferation by agents such as alpha-adrenergic receptor agonists. Nevertheless, how the CaMKII and ERK pathways interact in VSMCs has yet to be characterized. The aim of the present study was to clarify this interaction in response to alpha(1)-adrenergic receptor-mediated VSMC proliferation. We discovered that phenylephrine stimulation resulted in complex formation between CaMKII and ERK in a manner that facilitated phosphorylation of both protein kinases. To assess the effects of CaMKII/ERK association on VSMC proliferation, we inhibited endogenous CaMKII either pharmacologically or by adenoviral-mediated gene transfer of a kinase-inactive CaMKII mutant. Inhibition of CaMKII activation but not CaMKII autonomous activity significantly decreased formation of the CaMKII/ERK complex. On the contrary, the expression of constitutively active CaMKII enhanced VSMC growth and CaMKII/ERK association. In addressing the mechanism of this effect, we found that CaMKII could not directly phosphorylate ERK but instead enhanced Raf1 activation. By contrast, ERK interaction with CaMKII facilitated CaMKII phosphorylation and promoted its nuclear localization. Our results reveal a critical role for CaMKII in VSMC proliferation and imply that CaMKII facilitates assembly of the Raf/MEK/ERK complex and that ERK enhances CaMKII activation and influences its subcellular localization.

Physiological roles of the Ca2+/CaM-dependent protein kinase cascade in health and disease.

Numerous hormones, growth factors and physiological processes cause a rise in cytosolic Ca2+, which is translated into meaningful cellular responses by interacting with a large number of Ca2(+)-binding proteins. The Ca2(+)-binding protein that is most pervasive in mediating these responses is calmodulin (CaM), which acts as a primary receptor for Ca2+ in all eukaryotic cells. In turn, Ca2+/CaM functions as an allosteric activator of a host of enzymatic proteins including a considerable number of protein kinases. The topic of this review is to discuss the physiological roles of a sub-set of these protein kinases which can function in cells as a Ca2+/CaM-dependent kinase signaling cascade. The cascade was originally believed to consist of a CaM kinase kinase that phosphorylates and activates one of two CaM kinases, CaMKI or CaMKIV. The unusual aspect of this cascade is that both the kinase kinase and the kinase require the binding of Ca2+/CaM for activation. More recently, one of the CaM kinase kinases has been found to activate another important enzyme, the AMP-dependent protein kinase so the concept of the CaM kinase cascade must be expanded. A CaM kinase cascade is important for many normal physiological processes that when misregulated can lead to a variety of disease states. These processes include: cell proliferation and apoptosis that may conspire in the genesis of cancer; neuronal growth and function related to brain development, synaptic plasticity as well as memory formation and maintenance; proper function of the immune system including the inflammatory response, activation of T lymphocytes and hematopoietic stem cell maintenance; and the central control of energy balance that, when altered, can lead to obesity and diabetes. Although the study of the CaM-dependent kinase cascades is still in its infancy continued analysis of the pathways regulated by these Ca2(+)-initiated signaling cascades holds considerable promise for the future of disease-related research.

Spermatogenesis and the regulation of Ca(2+)-calmodulin-dependent protein kinase IV localization are not dependent on calspermin.

Calspermin and Ca(2+)-calmodulin-dependent protein kinase IV (CaMKIV) are two proteins encoded by the Camk4 gene. CaMKIV is found in multiple tissues, including brain, thymus, and testis, while calspermin is restricted to the testis. In the mouse testis, both proteins are expressed within elongating spermatids. We have recently shown that deletion of CaMKIV has no effect on calspermin expression but does impair spermiogenesis by disrupting the exchange of sperm basic nuclear proteins. The function of calspermin within the testis is unclear, although it has been speculated to play a role in binding and sequestering calmodulin during the development of the germ cell. To investigate the contribution of calspermin to spermatogenesis, we have used Cre/lox technology to specifically delete calspermin, while leaving kinase expression intact. We unexpectedly found that calspermin is not required for male fertility. We further demonstrate that CaMKIV expression and localization are unaffected by the absence of calspermin and that calspermin does not colocalize to the nuclear matrix with CaMKIV.

Expression of Ca(2+)/calmodulin-dependent protein kinase IV (caMKIV) messenger RNA during murine embryogenesis.

Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV) is a monomeric, multifunctional serine/threonine protein kinase that is expressed in subanatomic regions of the central and peripheral nervous system, T lymphocytes, and male germ cells. It is frequently localized to the nucleus, where it serves as a mediator of Ca(2+)-dependent gene expression. Although CaMKIV expression in the adult rat central nervous system and thymus has been described, little is known about the embryonic expression of murine CaMKIV. Here we report a thorough embryonic expression study of CaMKIV mRNA from embryonic day 9.5 through postnatal day 1. Expression patterns during embryonic development are significantly different from those of adults, suggesting specific roles for CaMKIV during development. Regions of high CaMKIV mRNA expression include thymic and bone cartilage primordia as well as specific cranial nerve ganglia (trigeminal, vestibulocochlear, and glossopharyngeal), thalamus, and dorsal root ganglia. This pattern of expression is chronologically consistent with periods of extensive cellular differentiation, proliferation, or neuronal survival selection and shows a predilection for neural crest-derived cells. These trends, along with recent studies in the CaMKIV null mouse, suggest that CaMKIV may play an important physiological role in cellular differentiation during embryogenesis.

The modular nature of histone deacetylase HDAC4 confers phosphorylation-dependent intracellular trafficking.

In C2C12 myoblasts, endogenous histone deacetylase HDAC4 shuttles between cytoplasmic and nuclear compartments, supporting the hypothesis that its subcellular localization is dynamically regulated. However, upon differentiation, this dynamic equilibrium is disturbed and we find that HDAC4 accumulates in the nuclei of myotubes, suggesting a positive role of nuclear HDAC4 in muscle differentiation. Consistent with the notion of regulation of HDAC4 intracellular trafficking, we reveal that HDAC4 contains a modular structure consisting of a C-terminal autonomous nuclear export domain, which, in conjunction with an internal regulatory domain responsive to calcium/calmodulin-dependent protein kinase IV (CaMKIV), determines its subcellular localization. CaMKIV phosphorylates HDAC4 in vitro and promotes its nuclear-cytoplasmic shuttling in vivo. However, although 14-3-3 binding of HDAC4 has been proposed to be important for its cytoplasmic retention, we find this interaction to be independent of CaMKIV. Rather, the HDAC4.14-3-3 complex exists in the nucleus and is required to confer CaMKIV responsiveness. Our results suggest that the subcellular localization of HDAC4 is regulated by sequential phosphorylation events. The first event is catalyzed by a yet to be identified protein kinase that promotes 14-3-3 binding, and the second event, involving protein kinases such as CaMKIV, leads to efficient nuclear export of the HDAC4.14-3-3 complex.

Ca(2+)/CaM-dependent kinases: from activation to function.

Calmodulin (CaM) is an essential protein that serves as a ubiquitous intracellular receptor for Ca(2+). The Ca(2+)/CaM complex initiates a plethora of signaling cascades that culminate in alteration of cellular functions. Among the many Ca(2+)/CaM-binding proteins to be discovered, the multifunctional protein kinases CaMKI, II, and IV play pivotal roles. Our review focuses on this class of CaM kinases to illustrate the structural and biochemical basis for Ca(2+)/CaM interaction with and regulation of its target enzymes. Gene transcription has been chosen as the functional endpoint to illustrate the recent advances in Ca(2+)/CaM-mediated signal transduction mechanisms.

Female fertility is reduced in mice lacking Ca2+/calmodulin-dependent protein kinase IV.

Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) is a serine/threonine protein kinase with limited tissue distribution. CaMKIV is highly expressed in the testis, where it is found in transcriptionally inactive elongating spermatids. We have recently generated mice deficient in CaMKIV. In the absence of CaMKIV, the exchange of sperm nuclear basic proteins in male spermatids is impaired, resulting in male infertility secondary to defective spermiogenesis. The involvement of CaMKIV in female fertility has not been addressed. Here we report that female fertility is markedly reduced in CaMKIV-deficient mice due to impaired follicular development and ovulation. CaMKIV is expressed in the ovary, where it is localized in granulosa cells. We further find that in cultured granulosa cells, CaMKIV expression and subcellular localization are hormonally regulated. As granulosa cells differentiate, CaMKIV levels decrease and the kinase translocates from the nucleus into the cytoplasm. Our results demonstrate a critical role for CaMKIV in female reproduction and point to a potential function in granulosa cell differentiation.

Cerebellar defects in Ca2+/calmodulin kinase IV-deficient mice.

The Ca(2+)/calmodulin-dependent protein kinase CaMKIV was first identified in the cerebellum and has been implicated in nuclear signaling events that control neuronal growth, differentiation, and plasticity. To understand the physiological importance of CaMKIV, we disrupted the mouse Camk4 gene. The CaMKIV null mice displayed locomotor defects consistent with altered cerebellar function. Although the overall cytoarchitecture of the cerebellum appeared normal in the Camk4(-/-) mice, we observed a significant reduction in the number of mature Purkinje neurons and reduced expression of the protein marker calbindin D28k within individual Purkinje neurons. Western immunoblot analyses of cerebellar extracts also established significant deficits in the phosphorylation of cAMP response element-binding protein at serine-133, a proposed target of CaMKIV. Additionally, the absence of CaMKIV markedly altered neurotransmission at excitatory synapses in Purkinje cells. Multiple innervation by climbing fibers and enhanced parallel fiber synaptic currents suggested an immature development of Purkinje cells in the Camk4(-/-) mice. Together, these findings demonstrate that CaMKIV plays key roles in the function and development of the cerebellum.

Identification and characterization of two Ca2+/CaM-dependent protein kinases required for normal nuclear division in Aspergillus nidulans.

We utilized an expression screen to identify two novel Ca(2+)/calmodulin (CaM)-regulated protein kinases in Aspergillus nidulans. The two kinases, CMKB and CMKC, possess high sequence identity with mammalian CaM kinases (CaMKs) I/IV and CaMKKalpha/beta, respectively. In vitro CMKC phosphorylates and increases the activity of CMKB, indicating they are biochemical homologues of CaMKKalpha/beta and CaMKI/IV. The disruption of CMKB is lethal; however, when protein expression is postponed, the spores germinate with delayed kinetics. The observed lag corresponds to a delay in the G(1)-phase activation of the cyclin-dependent kinase NIMX(cdc2). Disruption of cmkC is not lethal, but spores lacking CMKC also germinate with delayed kinetics and a lag in the activation of NIMX(cdc2). Analysis of DeltacmkC suggests a role for CMKC in regulating the first and subsequent nuclear division cycles. We conclude that both CMKB and CMKC are required for the proper temporal activation of NIMX(cdc2) as spores enter the cell cycle from quiescence and suggest that this relationship exists during the G(1)/S transition of subsequent cell divisions.

Spermiogenesis and exchange of basic nuclear proteins are impaired in male germ cells lacking Camk4.

Ca2+/calmodulin-dependent protein kinase IV (Camk4; also known as CaMKIV), a multifunctional serine/threonine protein kinase with limited tissue distribution, has been implicated in transcriptional regulation in lymphocytes, neurons and male germ cells. In the mouse testis, however, Camk4 is expressed in spermatids and associated with chromatin and nuclear matrix. Elongating spermatids are not transcriptionally active, raising the possibility that Camk4 has a novel function in male germ cells. To investigate the role of Camk4 in spermatogenesis, we have generated mice with a targeted deletion of the gene Camk4. Male Camk4-/- mice are infertile with impairment of spermiogenesis in late elongating spermatids. The sequential deposition of sperm basic nuclear proteins on chromatin is disrupted, with a specific loss of protamine-2 and prolonged retention of transition protein-2 (Tnp2) in step-15 spermatids. Protamine-2 is phosphorylated by Camk4 in vitro, implicating a connection between Camk4 signalling and the exchange of basic nuclear proteins in mammalian male germ cells. Defects in protamine-2 have been identified in sperm of infertile men, suggesting that our results may have clinical implications for the understanding of human male infertility.

Chronic elevation of calmodulin in the ventricles of transgenic mice increases the autonomous activity of calmodulin-dependent protein kinase II, which regulates atrial natriuretic factor gene expression.

Although isoforms of Ca2+/calmodulin-dependent protein kinase II (CaMKII) have been implicated in the regulation of gene expression in cultured cells, this issue has yet to be addressed in vivo. We report that the overexpression of calmodulin in ventricular myocytes of transgenic mice results in an increase in the Ca2+/calmodulin-independent activity of endogenous CaMKII. The calmodulin transgene is regulated by a 500-bp fragment of the atrial natriuretic factor (ANF) gene promoter which, based on cell transfection studies, is itself known to be regulated by CaMKII. The increased autonomous activity of CaMKII maintains the activity of the transgene and establishes a positive feed-forward loop, which also extends the temporal expression of the endogenous ANF promoter in ventricular myocytes. Both the increased activity of CaMKII and transcriptional activation of ANF are highly selective responses to the chronic overexpression of calmodulin. These results indicate that CaMKII can regulate gene expression in vivo and suggest that this enzyme may represent the Ca2+-dependent target responsible for reactivation of the ANF gene during ventricular hypertrophy.

Calmodulin: a prototypical calcium sensor.

Calmodulin is the best studied and prototypical example of the E-F-hand family of Ca2+-sensing proteins. Changes in intracellular Ca2+ concentration regulate calmodulin in three distinct ways. First, at the cellular level, by directing its subcellular distribution. Second, at the molecular level, by promoting different modes of association with many target proteins. Third, by directing a variety of conformational states in calmodulin that result in target-specific activation. The calmodulin-dependent regulation of protein kinases illustrates the potential mechanisms by which Ca2+-sensing proteins can recognize and generate affinity and specificity for effectors in a Ca2+-dependent manner.

Requirement of the prolyl isomerase Pin1 for the replication checkpoint.

The peptidyl-prolyl isomerase Pin1 has been implicated in regulating cell cycle progression. Pin1 was found to be required for the DNA replication checkpoint in Xenopus laevis. Egg extracts depleted of Pin1 inappropriately transited from the G2 to the M phase of the cell cycle in the presence of the DNA replication inhibitor aphidicolin. This defect in replication checkpoint function was reversed after the addition of recombinant wild-type Pin1, but not an isomerase-inactive mutant, to the depleted extract. Premature mitotic entry in the absence of Pin1 was accompanied by hyperphosphorylation of Cdc25, activation of Cdc2/cyclin B, and generation of epitopes recognized by the mitotic phosphoprotein antibody, MPM-2. Therefore, Pin1 appears to be required for the checkpoint delaying the onset of mitosis in response to incomplete replication.

Ca(2+)/calmodulin-dependent protein kinase IV is expressed in spermatids and targeted to chromatin and the nuclear matrix.

Ca(2+)/calmodulin-dependent protein kinase IV and calspermin are two proteins encoded by the Camk4 gene. Both are highly expressed in the testis, where in situ hybridization studies in rat testes have demonstrated that CaMKIV mRNA is localized to pachytene spermatocytes, while calspermin mRNA is restricted to spermatids. We have examined the expression patterns of both CaMKIV and calspermin in mouse testis and unexpectedly find that CaMKIV is expressed in spermatogonia and spermatids but excluded from spermatocytes, while calspermin is found only in spermatids. CaMKIV and calspermin expression in the testis are stage-dependent and appear to be coordinately regulated. In germ cells, we find that CaMKIV is associated with the chromatin. We further demonstrate that a fraction of CaMKIV in spermatids is hyperphosphorylated and specifically localized to the nuclear matrix. These novel findings may implicate CaMKIV in chromatin remodeling during nuclear condensation of spermatids.

Activation of orphan receptor-mediated transcription by Ca(2+)/calmodulin-dependent protein kinase IV.

Retinoid-related receptor alpha (RORalpha) is an orphan nuclear receptor that constitutively activates transcription from its cognate response element. We show that RORalpha is Ca(2+ )responsive, and a Ca(2+)/calmodulin-independent form of Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV) potentiates RORalpha-dependent transcription 20- to 30-fold. Other orphan receptors including RORalpha2, RORgamma and COUP-TFI are also potentiated by CaMKIV. Transcriptional activation by CaMKIV is orphan receptor selective and does not occur with either the thyroid hormone or estrogen receptor. CaMKIV does not phosphorylate RORalpha or its ligand-binding domain (LBD) in vitro, although the LBD is essential for transactivation. Therefore, the RORalpha LBD was used in the mammalian two-hybrid assay to identify a single class of small peptide molecules containing LXXLL motifs that interacted with greater affinity in the presence of CaMKIV. This class of peptides antagonized activation of orphan receptor-mediated transcription by CaMKIV. These studies demonstrate a pivotal role for CaMKIV in the regulation of orphan receptor-mediated transcription.

c-Jun enhancement of cyclic adenosine 3',5'-monophosphate response element-dependent transcription induced by transforming growth factor-beta is independent of c-Jun binding to DNA.

Transforming growth factor-beta (TGFbeta) enhances transcription from reporter genes regulated by a single consensus cAMP-response element (CRE) upon transfection into the immortalized human keratinocyte cell line, HaCaT. Whereas both CRE-binding protein (CREB) and c-Jun present in extracts of unstimulated cells can complex with a CRE in gel-shift experiments, TGFbeta treatment increases the amount of c-Jun found in the complex. Overexpression of c-Jun is sufficient to increase CRE and GAL4-CREB-dependent transcription and mimics the stimulatory effects of TGFbeta on transcription from either reporter gene. Surprisingly, although a portion of CREB in unstimulated cells is phosphorylated on the activating serine residue, Ser-133, this level of phospho-CREB is not altered by TGFbeta treatment. In fact, the CREB-dependent transcriptional effects of TGFbeta or c-Jun do not require phosphorylation of Ser-133, although CREB-binding protein (CBP) is required as evidenced by the observation that the adenoviral oncoprotein E1A can block the effects of both agents. c-Jun enhancement of CRE or GAL4-CREB-dependent transcription neither requires the DNA-binding nor N-terminal domains of c-Jun. Collectively, these results are consistent with a model in which signaling pathways initiated by TGFbeta can stimulate CREB-dependent transcription by increasing the cellular concentration of c-Jun, which participates in activation of the CBP-containing transcription complex.

Calmodulin kinase II chimeras used to investigate the structural requirements for smooth muscle myosin light chain kinase autoinhibition and calmodulin-dependent activation.

Segments of the autoregulatory domain of MK, a catalytically active fragment of the monomeric smooth muscle myosin light chain kinase (smMLCK) (residues 472-972), were replaced with their counterparts from a homologous but multimeric enzyme, calmodulin-dependent protein kinase II (CaM KII). Chimeric proteins in which both the autoregulatory and oligomerization domains of CaM KII (residues 281-478) were substituted for residues 781-972 of smMLCK, MK(CK281-478), or only the autoregulatory domain of CaM KII (residues 281-315) was exchanged for residues 781-813 of smMLCK, MK(CK281-315), exhibited significant enzymatic activity in the absence of Ca(2+)/CaM. In contrast, both MK and a chimeric protein in which the C-terminal half of the autoregulatory domain of smMLCK was replaced with CaM KII residues 301-315, MK(CK301-315), were inactive in the absence of Ca(2+)/CaM. These results indicate that the sequence of the N-terminal half of the autoregulatory domain of smMLCK is important for complete autoinhibition of its enzymatic activity. All proteins bound to Ca(2+)/CaM, and the chimeric proteins MK(CK281-478) and MK(CK281-315) were activated by Ca(2+)/CaM with activation constants (K(CaM)) and maximal enzymatic activities comparable to those of the wild-type MK enzyme. This demonstrates that the entire autoregulatory domain of CaM KII can replace that of smMLCK in its ability to promote efficient CaM-dependent activation of the smMLCK enzyme. However, the inability of the chimeric protein MK(CK301-315) to be activated by Ca(2+)/CaM suggests that replacement of only the C-terminal half of the autoregulatory domain of smMLCK, while still retaining the ability to bind Ca(2+)/CaM, also substitutes residues that prevent activation of the enzyme by Ca(2+)/CaM.

Modulation of Xenopus oocyte-expressed phospholemman-induced ion currents by co-expression of protein kinases.

Phospholemman (PLM), the major sarcolemmal substrate for phosphorylation by cAMP-dependent kinase (PKA) protein kinase C (PKC) and NIMA kinase in muscle, induces hyperpolarization-activated anion currents in Xenopus oocytes, most probably by enhancing endogenous oocyte currents. PLM peptides from the cytoplasmic tail are phosphorylated by PKA at S68, by NIMA kinase at S63, and by PKC at both S63 and S68. We have confirmed the phosphorylation sites in the intact protein, and we have investigated the role of phosphorylation in the regulatory activity of PLM using oocyte expression experiments. We found: (1) the cytoplasmic domain is not essential for inducing currents in oocytes; (2) co-expression of PKA increased the amplitude of oocyte currents and the amount of PLM in the oocyte membrane largely, but not exclusively, through phosphorylation of S68; (3) co-expression of PKA had no effect on a PLM mutant in which all putative phosphorylation sites had been inactivated by serine to alanine mutation (SSST 62, 63, 68, 69 AAAA); (4) co-expression of PKC had no effect in this system; (5) co-expression of NIMA kinase increased current amplitude and membrane protein level, but did not require PLM phosphorylation. These findings point to a role for phosphorylation in the function of PLM.