Subcellular distributions of rat CaM kinase phosphatase N and other members of the CaM kinase regulatory system

Ca2+/Calmodulin-dependent protein kinase (CaM kinase) regulatory system is composed of multifunctional CaM kinases such as CaM kinases IV and I, upstream CaM kinases such as CaM kinase kinases alpha and beta, which activate multifunctional CaM kinases, and CaM kinase phosphatases such as CaM kinase phosphatase and CaM kinase phosphatase N, which deactivate the activated multifunctional CaM kinases. To understand the combinations of CaM kinases I and IV, CaM kinase kinases alpha and beta, and CaM kinase phosphatases, the locations of the enzymes in the cell were examined by immunocytochemical studies of cultured cells. The results indicate that CaM kinase I, CaM kinase kinase beta, and CaM kinase phosphatase occur in the cytoplasm and that CaM kinase IV, CaM kinase kinase alpha (and CaM kinase kinase beta in some cell types and tissues), and CaM kinase phosphatase N occur inside the cellular nucleus, suggesting that there are at least two different sets of CaM kinase regulatory systems, one consisting of CaM kinase I, CaM kinase kinase beta, and CaM kinase phosphatase in the cytoplasm and the other consisting of CaM kinase IV, CaM kinase kinase alpha (and CaM kinase kinase beta in some cell types and tissues), and CaM kinase phosphatase N in the nucleus.     

Calcium/calmodulin-dependent phosphorylation and activation of human Cdc25-C at the G2/M phase transition in HeLa cells

The human tyrosine phosphatase (p54(cdc25-c)) is activated by phosphorylation at mitosis entry. The phosphorylated p54(cdc25-c) in turn activates the p34-cyclin B protein kinase and triggers mitosis. Although the active p34-cyclin B protein kinase can itself phosphorylate and activate p54(cdc25-c), we have investigated the possibility that other kinases may initially trigger the phosphorylation and activation of p54(cdc25-c). We have examined the effects of the calcium/calmodulin-dependent protein kinase (CaM kinase II) on p54(cdc25-c). Our in vitro experiments show that CaM kinase II can phosphorylate p54(cdc25-c) and increase its phosphatase activity by 2.5-3-fold. Treatment of a synchronous population of HeLa cells with KN-93 (a water-soluble inhibitor of CaM kinase II) or the microinjection of AC3-I (a specific peptide inhibitor of CaM kinase II) results in a cell cycle block in G2 phase. In the KN-93-arrested cells, p54(cdc25-c) is not phosphorylated, p34(cdc2) remains tyrosine phosphorylated, and there is no increase in histone H1 kinase activity. Our data suggest that a calcium-calmodulin-dependent step may be involved in the initial activation of p54(cdc25-c).     

Inhibition of G2/M progression in Schizosaccharomyces pombe by a mutant calmodulin kinase II with constitutive activity.

Intracellular signaling by the second messenger Ca2+ through its receptor calmodulin (CaM) regulates cell function via the activation of CaM-dependent enzymes. Previous studies have shown that cell cycle progression at G1/S and G2/M is sensitive to intracellular CaM levels. However, little is known about the CaM-regulated enzymes involved. Protein phosphorylation has been shown to be important for cell-cycle regulation. Because CaM regulates several protein kinases, and at least one protein phosphatase, our studies are focusing on the roles of these enzymes within the cell cycle. As an initial approach to this problem, cDNAs encoding either normal or mutant calcium/calmodulin kinase II (CaMKII) have been expressed in Schizosaccharomyces pombe. The results show that overexpression of a constitutively active mutant CaMKII caused cell-cycle arrest in G2. Arrest was associated with a failure to activate the p34/cdc2 protein kinase. Expression of the mutant CaMKII in strains of S. pombe with altered timing of mitosis revealed that this effect is not mediated either by cdc25+ or wee1+, suggesting that CaMKII may regulate G2/M progression by another mechanism.     

Extragenic suppressors of the nimX2(cdc2) mutation of Aspergillus nidulans affect nuclear division, septation and conidiation

The Aspergillus nidulans NIMX(CDC2) protein kinase has been shown to be required for both the G(2)/M and G(1)/S transitions, and recent evidence has implicated a role for NIMX(CDC2) in septation and conidiation. While much is understood of its G(2)/M function, little is known about the functions of NIMX(CDC2) during G(1)/S, septation, and conidiophore development. In an attempt to better understand how NIMX(CDC2) is involved in these processes, we have isolated four extragenic suppressors of the A. nidulans nimX2(cdc2) temperature-sensitive mutation. Mutation of these suppressor genes, designated snxA-snxD for suppressor of nimX, affects nuclear division, septation, and conidiation. The cold-sensitive snxA1 mutation leads to arrest of nuclear division during G(1) or early S. snxB1 causes hyperseptation in the hyphae and sensitivity to hydroxyurea, while snxC1 causes septation in the conidiophore stalk and aberrant conidiophore structure. snxD1 leads to slight septation defects and hydroxyurea sensitivity. The additional phenotypes that result from the suppressor mutations provide genetic evidence that NIMX(CDC2) affects septation and conidiation in addition to nuclear division, and cloning and biochemical analysis of these will allow a better understanding of the role of NIMX(CDC2) in these processes.     

TGFbeta regulates the expression and activities of G2 checkpoint kinases in human myeloid leukemia cells

Transforming Growth Factor-beta (TGFbeta) is known to be a negative regulator of G1 cyclin/cdk activity. It is not clear whether TGFbeta has any effect on G2 checkpoint kinases. We have found that TGFbeta downregulated the expression of several G2 checkpoint kinases including cdc2, cyclin B1, and cdc25c without causing cell accumulation in G2/M phases in two human leukemia cell lines. The inhibition was time-dependent with a maximal inhibition being observed by 24h for cyclin B1 and cdc2 and by 48h for cdc25c. The inhibition was not a result of G1 arrest but a direct effect of TGFbeta which downregulates their expression at mRNA level. In proliferating cells, there was a significant formation of cdc2-pRb complexes, which was decreased to 30% of control levels by 48h after initiating TGFbeta treatment. Cdc2 showed a marked kinase activity on GST-Rb protein in proliferating cells detected by in vitro kinase assay, which was downregulated in response to TGFbeta. In addition, TGFbeta caused a rapid and transient dephosphorylation of cdc2 (Tyr15) and cdc25c (Ser216) for about 2-3h before a dramatic decrease of both molecules by 48h. Taken together, our data suggest that TGFbeta has a direct inhibitory effect on G2 checkpoint kinases, which is regulated at mRNA level. The transient activation of cdc2 and cdc25c and subsequent inhibition of cdc2, cyclin B1, and cdc25c could amplify TGFbeta-induced G1 arrest and growth inhibition.     

Activation of Ca2+/calmodulin-dependent protein kinase I in cultured rat hippocampal neurons

We have focused on activation mechanisms of calcium/calmodulin-dependent protein kinase (CaM) kinase I in the hippocampal neurons and compared them with that of CaM kinase IV. Increased activation of CaM kinase I occurred by stimulation with glutamate and depolarization in cultured rat hippocampal neurons. Similar to CaM kinases II and IV, CaM kinase I was essentially activated by stimulation with the NMDA receptor. Although both CaM kinases I and IV seem to be activated by CaM kinase kinase, the activation of CaM kinase I was persistent during stimulation with glutamate in contrast to a transient activation of CaM kinase IV. In addition, CaM kinase I was activated in a lower concentration of glutamate than that of CaM kinase IV. Depolarization-induced activation of CaM kinase I was also evident in the cultured neurons and was largely blocked by nifedipine. In the experiment with 32P-labeled cells, phosphorylation of CaM kinase I was stimulated by glutamate treatment and depolarization. The glutamate- and depolarization-induced phosphorylation was inhibited by the NMDA receptor antagonist and nifedipine, respectively. These results suggest that, although CaM kinases I and IV are activated by the NMDA receptor and depolarization stimulation, these kinase activities are differently regulated in the hippocampal neurons.     

Redox regulation of the calcium/calmodulin-dependent protein kinases

Reactive oxygen intermediates (ROI) have been viewed traditionally as damaging to the cell. However, a predominance of evidence has shown that ROI can also function as important activators of key cellular processes, and ROI have been shown to play a vital role in cell signaling networks. The calcium/calmodulin-dependent protein kinases (CaM kinases) are a family of related kinases that are activated in response to increased intracellular calcium concentrations. In this report we demonstrate that hydrogen peroxide treatment results in the activation of both CaM kinase II and IV in Jurkat T lymphocytes. Surprisingly, this activation occurs in the absence of any detectable calcium flux, suggesting a novel means for the activation of these kinases. Treatment of Jurkat cells with phorbol 12-myristate 13-acetate (PMA), which does not cause a calcium flux, also activated the CaM kinases. The addition of catalase to the cultures inhibited PMA-induced activation of the CaM kinases, suggesting that similar to hydrogen peroxide, PMA also activates the CaM kinases via the production of ROI. One mechanism by which this likely occurs is through oxidation and consequential inactivation of cellular phosphatases. In support of this concept, okadaic acid and microcystin-LR, which are inhibitors of protein phosphatase 2A (PP2A), induced CaM kinase II and IV activity in these cells. Overall, these results demonstrate a novel mechanism by which ROI can induce CaM kinase activation in T lymphocytes.     

The G2/M DNA damage checkpoint inhibits mitosis through Tyr15 phosphorylation of p34cdc2 in Aspergillus nidulans.

It is possible to cause G2 arrest in Aspergillus nidulans by inactivating either p34cdc2 or NIMA. We therefore investigated the negative control of these two mitosis-promoting kinases after DNA damage. DNA damage caused rapid Tyr15 phosphorylation of p34cdc2 and transient cell cycle arrest but had little effect on the activity of NIMA. Dividing cells deficient in Tyr15 phosphorylation of p34cdc2 were sensitive to both MMS and UV irradiation and entered lethal premature mitosis with damaged DNA. However, non-dividing quiescent conidiospores of the Tyr15 mutant strain were not sensitive to DNA damage. The UV and MMS sensitivity of cells unable to tyrosine phosphorylate p34cdc2 is therefore caused by defects in DNA damage checkpoint regulation over mitosis. Both the nimA5 and nimT23 temperature-sensitive mutations cause an arrest in G2 at 42 degrees C. Addition of MMS to nimT23 G2-arrested cells caused a marked delay in their entry into mitosis upon downshift to 32 degrees C and this delay was correlated with a long delay in the dephosphorylation and activation of p34cdc2. Addition of MMS to nimA5 G2-arrested cells caused inactivation of the H1 kinase activity of p34cdc2 due to an increase in its Tyr15 phosphorylation level and delayed entry into mitosis upon return to 32 degrees C. However, if Tyr15 phosphorylation of p34cdc2 was prevented then its H1 kinase activity was not inactivated upon MMS addition to nimA5 G2-arrested cells and they rapidly progressed into a lethal mitosis upon release to 32 degrees C. Thus, Tyr15 phosphorylation of p34cdc2 in G2 arrests initiation of mitosis after DNA damage in A. nidulans.     

Detecting protein kinase recognition modes of calmodulin by residual dipolar couplings in solution NMR

Calmodulin-regulated serine/threonine kinases (CaM kinases) play crucial roles in Ca2+-dependent signaling transduction pathways in eukaryotes. Despite having a similar overall molecular architecture of catalytic and regulatory domains, CaM kinases employ different binding modes for Ca2+/CaM recruitment which is required for their activation. Here we present a residual dipolar coupling (RDC)-based NMR approach to characterizing the molecular recognition of CaM with five different CaM kinases. Our analyses indicate that CaM kinase I and likely IV use the same CaM binding mode as myosin light chain kinase (1-14 motif), distinct from those of CaM kinase II (1-10 motif) and CaM kinase kinase (1-16- motif). This NMR approach provides an efficient experimental guide for homology modeling and structural characterization of CaM-target complexes.     

The human polo-like kinase, PLK, regulates cdc2/cyclin B through phosphorylation and activation of the cdc25C phosphatase

Entry into mitosis by mammalian cells is triggered by the activation of the cdc2/cyclin B holoenzyme. This is accomplished by the specific dephosphorylation of key residues by the cdc25C phosphatase. The polo-like kinases are a family of serine/threonine kinases which are also implicated in the control of mitotic events, but their exact regulatory mechanism is not known. Recently, a Xenopus homologue, PLX1, was reported to phosphorylate and activate cdc25, leading to activation of cdc2/cyclin B. Jurkat T leukemia cells were chemically arrested and used to verify that PLK protein expression and its phosphorylation state is regulated with respect to cell cycle phase (i.e., protein is undetectable at G1/S, accumulates at S phase and is modified at G2/M). Herein, we show for the first time that endogenous human PLK protein immunoprecipitated from the G2/M-arrested Jurkat cells directly phosphorylates human cdc25C. In addition, we demonstrate that recombinant human (rh) PLK also phosphorylates rhcdc25C in a time- and concentration-dependent manner. Phosphorylation of endogenous cdc25C and recombinant cdc25C by PLK resulted in the activation of the phosphatase as assessed by dephosphorylation of cdc2/cyclin B. These data are the first to demonstrate that human PLK is capable of phosphorylating and positively regulating human cdc25C activity, allowing cdc25C to dephosphorylate inactive cdc2/cyclin B. As this event is required for cell cycle progression, we define at least one key regulatory mode of action for human PLK in the initiation of mitosis.     

Ca(2+)-calmodulin-dependent protein kinases Ia and Ib from rat brain. II. Enzymatic characteristics and regulation of activities by phosphorylation and dephosphorylation.

In addition to physical properties (DeRemer, M. F., Saeli, R. J., and Edelman, A. M. (1992) J. Biol. Chem. 267, 13460-13465), enzymatic and regulatory characteristics indicate that calmodulin (CaM) kinase Ia and CaM kinase Ib are distinct entities. The Km values for ATP and site 1 peptide were similar between the two kinases, however, CaM kinase Ib is approximately 20-fold more sensitive to CaM than is CaM kinase Ia. The kinases also displayed differential sensitivities to divalent metal ions. For both kinases, site 1 peptide, synapsin I, and syntide-2 were highly preferred substrates relative to others tested. A 72-kDa protein from a heat-treated extract of rat pancreas was phosphorylated by CaM kinase Ib but not by CaM kinase Ia. CaM kinase Ia activity displayed a pronounced lag in its time course suggesting enzyme activation over time. Preincubation of CaM kinase Ia in the combined presence of Ca(2+)-CaM and MgATP led to a time-dependent increase in its site 1 peptide kinase activity of up to 15-fold. The extent of activation of CaM kinase Ia correlated with the extent of autophosphorylation. The enzyme retained full Ca(2+)-CaM dependence in the activated state which was rapidly reversible by treatment with protein phosphatase 2A catalytic subunit. Thus, the activation of CaM kinase Ia is a result of its Ca(2+)-CaM-dependent autophosphorylation. CaM kinase Ib was not activated by preincubation under autophosphorylating conditions yet lost approximately 90% of its activity toward either an exogenous substrate (site 1 peptide) or itself (autophosphorylation) after incubation with protein phosphatase 2A catalytic subunit. The deactivated state was not reversed by subsequent incubations under autophosphorylating conditions. Thus, CaM kinase Ib activity is dependent upon phosphorylation by a regulating kinase(s) which is resolved from CaM kinase Ib during purification of the latter.     

Regulation of Saccharomyces Rad53 checkpoint kinase during adaptation from DNA damage-induced G2/M arrest

Saccharomyces cells with one unrepaired double-strand break (DSB) adapt after checkpoint-mediated G2/M arrest. Adaptation is accompanied by loss of Rad53p checkpoint kinase activity and Chk1p phosphorylation. Rad53p kinase remains elevated in yku70delta and cdc5-ad cells that fail to adapt. Permanent G2/M arrest in cells with increased single-stranded DNA is suppressed by the rfa1-t11 mutation, but this RPA mutation does not suppress permanent arrest in cdc5-ad cells. Checkpoint kinase activation and inactivation can be followed in G2-arrested cells, but there is no kinase activation in G1-arrested cells. We conclude that activation of the checkpoint kinases in response to a single DNA break is cell cycle regulated and that adaptation is an active process by which these kinases are inactivated.     

The NIMA protein kinase is hyperphosphorylated and activated downstream of p34cdc2/cyclin B: coordination of two mitosis promoting kinases.

Initiation of mitosis in Aspergillus nidulans requires activation of two protein kinases, p34cdc2/cyclin B and NIMA. Forced expression of NIMA, even when p34cdc2 was inactivated, promoted chromatin condensation. NIMA may therefore directly cause mitotic chromosome condensation. However, the mitosis-promoting function of NIMA is normally under control of p34cdc2/cyclin B as the active G2 form of NIMA is hyperphosphorylated and further activated by p34cdc2/cyclin B when cells initiate mitosis. To see the p34cdc2/cyclin B dependent activation of NIMA, okadaic acid had to be added to isolation buffers to prevent dephosphorylation of NIMA during isolation. Hyperphosphorylated NIMA contained the MPM-2 epitope and, in vitro, phosphorylation of NIMA by p34cdc2/cyclin B generated the MPM-2 epitope, suggesting that NIMA is phosphorylated directly by p34cdc2/cyclin B during mitotic initiation. These two kinases, which are both essential for mitotic initiation, are therefore independently activated as protein kinases during G2. Then, to initiate mitosis, we suggest that each activates the other's mitosis-promoting functions. This ensures that cells coordinately activate p34cdc2/cyclin B and NIMA to initiate mitosis only upon completion of all interphase events. Finally, we show that NIMA is regulated through the cell cycle like cyclin B, as it accumulates during G2 and is degraded only when cells traverse mitosis.     

Differential effects of a calcineurin inhibitor on glutamate-induced phosphorylation of Ca2+/calmodulin-dependent protein kinases in cultured rat hippocampal neurons

Calcium/calmodulin-dependent protein kinases (CaM kinases) are major multifunctional enzymes that play important roles in calcium-mediated signal transduction. To characterize their regulatory mechanisms in neurons, we compared glutamate-induced phosphorylation of CaM kinase IV and CaM kinase II in cultured rat hippocampal neurons. We observed that dephosphorylation of these kinases followed different time courses, suggesting different regulatory mechanisms for each kinase. Okadaic acid, an inhibitor of protein phosphatase (PP) 1 and PP2A, increased the phosphorylation of both kinases. In contrast, cyclosporin A, an inhibitor of calcineurin, showed different effects: the phosphorylation and activity of CaM kinase IV were significantly increased with this inhibitor, but those of CaM kinase II were not significantly increased. Cyclosporin A treatment of neurons increased phosphorylation of Thr196 of CaM kinase IV, the activated form with CaM kinase kinase, which was recognized with an anti-phospho-Thr196 antibody. Moreover, recombinant CaM kinase IV was dephosphorylated and inactivated with calcineurin as well as with PP1, PP2A, and PP2C in vitro. These results suggest that CaM kinase IV, but not CaM kinase II, is directly regulated with calcineurin.     

Germination of individual Bacillus subtilis spores with alterations in the GerD and SpoVA proteins, which are important in spore germination

Release of Ca(2+) with dipicolinic acid (CaDPA) was monitored by Raman spectroscopy and differential interference contrast microscopy during germination of individual spores of Bacillus subtilis strains with alterations in GerD and SpoVA proteins. Notable conclusions about germination after the addition of nutrient were as follows. (i) Following L-alanine addition, wild-type and gerD spores and spores with elevated SpoVA protein levels (↑SpoVA spores) slowly released ～10% of their CaDPA during a variable (6- to 55-min) period ending at T(lag), the time when faster CaDPA release began. (ii) T(lag) times were lower for ↑SpoVA spores than for wild-type spores and were higher for gerD spores. (iii) The long T(lag) times of gerD spores were partially due to slow commitment to germinate. (iv) The intervals between the commitment to germinate and CaDPA release were similar for wild-type and ↑SpoVA spores but longer for gerD spores. (v) The times for rapid CaDPA release, ΔT(release) = T(release) - T(lag) (with T(release) being the time at which CaDPA release was complete), were similar for wild-type, gerD, and ↑SpoVA spores. (vi) Spores with either one of two point mutations in the spoVA operon (spoVA(1) and spoVA(2) spores) exhibited a more rapid rate of CaDPA release beginning immediately after L-alanine addition leading to ～65% CaDPA release prior to T(lag). (vii) T(lag) times for spoVA(1) and spoVA(2) spores were longer than for wild-type spores. (viii) The intervals between spoVA(1) and spoVA(2) spores' commitment and CaDPA release were similar to those for wild-type spores, but commitment occurred later. In contrast to germination after the addition of nutrient, T(lag) and ΔT(release) times were relatively similar during dodecylamine germination of spores of the five strains. These findings suggest the following. (i) GerD plays no role in CaDPA release during spore germination. (ii) SpoVA proteins are involved in CaDPA release during germination with nutrients, and probably with dodecylamine. (iii) Spores release significant CaDPA before commitment. (iv) CaDPA release during T(lag) and ΔT(release) may signal subsequent germination events.     

Cyclin A- and cyclin B-dependent protein kinases are regulated by different mechanisms in Xenopus egg extracts.

Cyclins are proteins which are synthesized and degraded in a cell cycle-dependent fashion and form integral regulatory subunits of protein kinase complexes involved in the regulation of the cell cycle. The best known catalytic subunit of a cyclin-dependent protein kinase complex is p34cdc2. In the cell, cyclins A and B are synthesized at different stages of the cell cycle and induce protein kinase activation with different kinetics. The kinetics of activation can be reproduced and studied in extracts of Xenopus eggs to which bacterially produced cyclins are added. In this paper we report that in egg extracts, both cyclin A and cyclin B associate with and activate the same catalytic subunit, p34cdc2. In addition, cyclin A binds a less abundant p33 protein kinase related to p34cdc2, the product of the cdk2/Eg1 gene. When complexed to cyclin B, p34cdc2 is subject to transient inhibition by tyrosine phosphorylation, producing a lag between the addition of cyclin and kinase activation. In contrast, p34cdc2 is only weakly tyrosine phosphorylated when bound to cyclin A and activates rapidly. This finding shows that a given kinase catalytic subunit can be regulated in a different manner depending on the nature of the regulatory subunit to which it binds. Tyrosine phosphorylation of p34cdc2 when complexed to cyclin B provides an inhibitory check on the activation of the M phase inducing protein kinase, allowing the coupling of processes such as DNA replication to the onset of metaphase. Our results suggest that, at least in the early Xenopus embryo, cyclin A-dependent protein kinases may not be subject to this checkpoint and are regulated primarily at the level of cyclin translation.