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PHAGOCYTES
From the Department of Pulmonary Diseases, University
Medical Centre Utrecht, 3584 CX Utrecht, The Netherlands
Human granulocytes are characterized by a variety of specific
effector functions involved in host defense. Several widely expressed
protein kinases have been implicated in the regulation of these
effector functions. A polymerase chain reaction-based strategy was
used to identify novel granulocyte-specific kinases. A novel protein
kinase complementary DNA with an open reading frame of 357 amino acids
was identified with homology to calcium-calmodulin-dependent kinase I
(CaMKI). This has been termed CaMKI-like kinase (CKLiK). Analysis of
CKLiK messenger RNA (mRNA) expression in hematopoietic cells
demonstrated an almost exclusive expression in human polymorphonuclear leukocytes (PMN). Up-regulation of CKLiK mRNA occurs during
neutrophilic differentiation of CD34+ stem cells. CKLiK
kinase activity was dependent on Ca++ and calmodulin as
analyzed by in vitro phosphorylation of cyclic adenosine monophosphate
responsive element modulator (CREM). Furthermore, CKLiK- transfected
cells treated with ionomycin demonstrated an induction of
CRE- binding protein (CREB) transcriptional activity compared
to control cells. Additionally, CaMK-kinase Human polymorphonuclear leukocytes (PMN), which
include neutrophilic and eosinophilic granulocytes, play an important
role in host defense against invading microorganisms.1
Recruitment and activation of these cells in vivo occur in a multistep
process that involves many different membrane-bound receptors
activating an array of diverse intracellular signaling molecules. In
short, PMNs in the peripheral blood enter a preactivated state by
interacting with cytokines liberated from the inflammatory locus. This
is followed by attachment to the endothelium, which is mediated by the
interaction with adhesion molecules expressed on the surface of
activated endothelial cells. Release of chemokines at the site of
inflammation is responsible for the migration of PMNs to this locus.
Finally on recognition of the inciting agent by PMNs, phagocytosis, secretion of toxic proteins, and activation of membrane-bound NADPH-oxidase generating reactive oxygen intermediates
ensues.2,3 Furthermore, rapid induction of apoptosis in
PMNs and subsequent removal of apoptotic cells are important in the
rapid resolution of inflammation.4,5 An unfortunate
consequence of activation in vivo is tissue damage during acute
inflammation and, therefore, the activity of granulocytes is under
tight control.
The PMNs express a wide variety of receptors on their plasma membranes,
steering the process of priming and activation. On binding of
inflammatory mediators, such as formyl peptides, lipopolysaccharides, chemokines, or cytokines, the receptor transmits a signal to the cell
interior resulting in the initiation of a cascade of intracellular events. Phosphorylation of effector molecules by kinases is critical for transducing intracellular signals. Thus far several classes of
kinases, including (1) serine kinases, such as mitogen activated (MAP)
kinases; (2) lipid kinases such as phosphatidylinositol-3 kinase
(PI-3K); (3) tyrosine kinases including the src kinases; (4) cyclic
adenosine monophosphate (cAMP)-dependent kinases, and (5)
Ca++-dependent kinases, are activated in response to
inflammatory mediators in human granulocytes.
A role for these widely expressed kinases in neutrophil functions has
been intensively studied using pharmacologic inhibitors. A role for MAP
kinases, p42ERK1 and p42ERK2, in chemotaxis,
respiratory burst, and platelet activating factor (PAF) release
is suggested, although the use of a pharmacologic inhibitor for
MAPK/ERK kinase (MEK) has resulted in contradictory findings.6,7 A clearer role for PI-3K in neutrophil
migration and respiratory burst has been demonstrated by use of PI-3K
inhibitors wortmannin and LY294002.7-11 A role for protein
kinase C (PKC) has also been postulated in a variety of granulocyte
effector functions and protein kinase A (PKA) is suggested to be
involved in down-regulating the respiratory burst.12,13
Recently a role for src kinases in adhesion-dependent degranulation has
also been described.14 Although inhibitory studies support
a role for these kinases in regulating neutrophil functions, their
activation is not specific for these granulocyte effector functions
because they are widely expressed. Furthermore, pharmacologic
inhibitors are often limited in their specificity, making the
interpretation of data more complex.15
Changes in cytosolic free Ca++ are described during
activation of several neutrophil responses, such as degranulation,
respiratory burst, and adhesion. An important role for Ca++
in these processes has been suggested16,17 and, therefore, Ca++-dependent kinases may well be involved. In addition a
role for the Ca++-dependent phosphatase, calcineurin has
been shown in the Ca++-dependent recycling of integrins to
the front of migrating neutrophils,18 whereas a role for
calmodulin and CaMKII is suggested in oxygen production.19
In this report we describe the identification of a novel protein
kinase, which we have termed CKLiK (CaMKI-like kinase). This kinase is
predominantly expressed in human PMNs and is regulated by
Ca++ and calmodulin. Interleukin (IL)-8, which is a potent
activator of neutrophil effector functions, induces activation of CKLiK and we show that an inducible active mutant of CKLiK induces ERK MAP
kinase activation. These data identify a novel
Ca++/calmodulin-dependent protein kinase expressed in PMNs
that may play a role in transducing chemokine-induced signals
regulating human granulocyte functions.
Cells, reagents, and antibodies
Identification and cloning of granulocyte kinases
Full-length cDNA was obtained by screening a Isolation of human leukocytes Blood was obtained from healthy volunteers at the donor service of the University Medical Centre (Utrecht, The Netherlands). Granulocytes were isolated from blood treated with 0.4% (wt/vol) trisodium citrate (pH 7.4) as previously described.23 Mononuclear cells were removed from granulocytes by centrifugation over isotonic Ficoll from Pharmacia (Uppsula, Sweden). After lysis of the erythrocytes in an isotonic NH4Cl solution, neutrophils were washed and resuspended in incubation buffer (20 mmol/L HEPES, 132 mmol/LM NaCl, 6 mmol/L KCl, 1 mmol/L MgSO4, 1.2 mmol/L KH2PO4, 5 mmol/L glucose, 1 mmol/L CaCl2). Granulocytes were incubated for 30 minutes at 37°C before experiments were performed. Monocytes were further separated from mononuclear lymphocytes by elutriation as described previously.24Isolation and differentiation of CD34+ stem cells The CD34+ cells were isolated using the MACS CD34+ progenitor cells isolation kit from Miltenyi Biotech (Auburn, CA). First, mononuclear cells were isolated from human umbilical cord blood cells by density centrifugation over a Ficoll solution. Cells were blocked and additionally incubated with a CD34 antibody in a phosphate buffer containing 2 mmol/L EDTA and 0.5% bovine serum albumin (BSA) (buffer M). Magnetic beads recognizing the CD34 antibody were added and the suspension was applied to an MS+ separation column placed in the magnetic cell separator MiniMACS. CD34 cells were allowed to pass through the
column by 3 wash steps with buffer M. CD34+ cells were
eluted with buffer M by removal of the column from the separator. To
obtain a pure CD34+ cell population the magnetic separation
step was repeated on a second column. After final elution stem cells
were counted and cultured. Before differentiation, CD34+
stem cells were proliferated for 1 week in the presence of TPO (10 ng/mL), SCF (50 ng/mL), FLT-3 ligand (50 ng/mL), and IL-6 (2 ng/mL). At
day 0 differentiation was started by addition of SCF, FLT-3 ligand,
IL-3 (10 ng/mL), and G-CSF (30 ng/mL). At days 3, 7, 10, 14, and 17, IL-3 and G-CSF were added and at day 21 only G-CSF was added to the
cells. At each point cells were counted, diluted to
0.5 × 106 cells/mL and, if possible, samples
were taken for mRNA isolation.
RNase protection Total mRNA from human monocytes, lymphocytes, granulocytes, and hematopoietic cell lines HL60, U937, and THP1 were isolated. In short, 108 cells were lysed in 2 mL GIT-C solution (6 mol/L guanidine thiocyanate, 25 mmol/L sodium citrate, 0.5% N'-lauroyl-sacosine, 100 mmol/L -mercaptoethanol), and RNAs were
further isolated by phenol extraction and ethanol precipitation.
32P-UTP-labeled antisense RNA transcript, corresponding to
the original PCR fragment coding the catalytic domain of CKLiK, was
generated using the Riboprobe in vitro transcription system (Promega)
and used as an RNA probe. As internal control a 90-bp antisense RNA probe of GAPDH was used. Total RNA samples (10 µg) were lyophilized and resuspended in 2 µL diethyl pyrocarbonate (DEPC) water.
Hybridization was performed with 105 cpm of each antisense
RNA probe in 25 µL 80% formamide, 40 mmol/L PIPES pH 6.4, 400 mmol/L
NaCl, and 1 mmol/L EDTA overnight at 45°C. Subsequently probes were
incubated for 1 hour in RNAse buffer (10 mmol/L Tris-Cl pH 7.5, 5 mmol/L EDTA, 300 mmol/L NaCl supplemented with 0.15 µL/mL T1 RNase)
at 37°C to degrade unhybridized RNA. The reaction was stopped by
addition of 10 µL proteinase K (5 mg/mL) and 10% sodium dodecyl
sulfate (SDS). Hybridized (double-stranded) RNA was purified by phenol
extraction and ethanol precipitation. The remaining pellet was
resuspended in 2 µL DEPC water and 2 µL RNA loading buffer (80%
formamide, 10 mmol/L EDTA, 1 mg/mL xylene cyanol, 1 mg/mL bromphenol
blue). Samples were heated for 5 minutes at 95°C and analyzed by
polyacrylamide gel electrophoresis (PAGE).
DNA constructs Epitope-tagged CKLiK was generated by PCR using the oligonucleotides forward XhoI; 5'-CCGCTCGAGTATGGCCCGGGAGAACGGC-3' and reverse KpnI; 5'-CCGGTACCCAAGTAG-CTGACATTACAGG-5') and ligated by XhoI/KpnI digest into pMT-HA. HA_CKLiK-309 and HA_CKLiK-296 were also generated by PCR using reverse primers introducing a stop codon at amino acid 310 or 297 309:5'-GCATTTCATGCTTGGCACCATT-3',296; 5'-GCTCTAGATCA-CTGGGCGCTGACGGACTC-3'). PCR products were cloned into pGEM-T vector and subcloned into pMT2-HA vector. Green fluorescent protein (GFP)-tagged CKLiK was generated by PCR and recloned in frame into pEGFP-C2 vector (Clontech Laboratories, Palo Alto, CA). HA_CKLiK was cloned from pMT-HA into pBabe25 by BamHI/ EcoRI digest. Untagged CKLiK-296 was obtained by PCR and cloned into pSG5.26 VSV-tagged CaMKK was generated by PCR on rat brain tissue cDNA using the
oligonucleotides 5'-CAGTCGACCAGGAATATCCACGGACTGA-3' and
5'-ATAGCGGCCGCC-GGATGCAGCCTCATCTTC-3' and cloned into the pMT2-VSV
vector. Tamoxifen-inducible CKLiK construct, ER_CKLiK-296, was
generated by PCR and cloned into the pCDNA3-ER-N vector. The constructs
for HA_PKB and HA_ERK have been previously described.27 CREB_GAL4, CREBS133A_GAL4, and GAL4CAT constructs were previously described.28
In vitro kinase assay The COS cells were transiently transfected with 10 µg of HA-tagged CKLiK-WT, CKLiK-309, or CKLiK-296 using calcium phosphate precipitation, and the medium was refreshed 16 hours later. For the CKLiK kinase assay, 24 hours later cells were stimulated with or without ionomycin for 5 minutes, washed twice with cold phosphate-buffered saline (PBS) and lysed in a buffer containing 1% NP-40, 20 mmol/L Tris-Cl pH 7.5, 150 mmol/L NaCl, 10% glycerol, and 10 mmol/L MgCl2 supplemented with 10 µg/mL aprotinin, 1 mmol/L leupeptin, 1 mmol/L PMSF, 1 mmol/L benzamidine, and 1 mmol/L Na3VO4. The 32D cells stably expressing CKLiK-WT were mIL-3 and serum starved for 4 hours and stimulated with hIL-8 (107 mol/L). Cells were washed and lysed in buffer
described above. Lysates were precleared for 20 minutes with protein-A
beads and immunoprecipitated with 12CA5 antibody. Immunoprecipitates
were washed twice in lysis buffer and twice in dilution buffer
containing 10 mmol/L Tris-Cl pH 7.4 and 20 mmol/L MgCl2.
Kinase assay was performed in the presence of 10 mmol/L Tris-Cl pH7.4,
20 mmol/L MgCl2, 1 mmol/L DTT, 50 µmol/L ATP, 0.1 µL
32P-dATP in the presence or absence of 1 mmol/L
CaCl2 and 0.5 µg calmodulin, 5 µg CREM (33 kd), or
mutated CREM -S117A (42 kd) as substrate.29 Protein
kinase B (PKB) and ERK kinase assay were performed as described
previously.27
CAT assay The COS cells were transiently transfected with 4 µg CKLiK-WT, -309, or -296, together with 2 µg CREB_GAL4 or CREB-S133A_GAL4 fusion expression plasmids and 2 µg GAL4CAT reporter construct using calcium phosphate precipitation. After 16 hours cells were washed twice and medium refreshed. Eight hours later cells were incubated overnight with 1 µmol/L ionomycin. Cells were lysed by repeated freeze-thawing in 100 µL 250 mmol/L Tris-Cl pH7.4 and 25 mmol/L EDTA. Then, 50µL of cellular extract was incubated in a total volume of 100 µL containing 250 mmol/L Tris-Cl 7.4, 2% glycerol, 0.3 mmol/L butyryl coenzyme A, and 0.05 µCi 14C-chloramphenicol for 2 hours at 37°C. Reaction products were extracted using 400 µL xylene/pristane 1:2 and the percentage of acetylated products was determined using liquid scintillation counting. A lacZ reporter was used to correct for transfection efficiency. Data represent at least 3 independent experiments ± SEM.Detection of GFP fusion-protein localization Plasmids encoding CKLiK and active CKLiK-296 containing N-terminal enhanced GFP were transiently transfected in COS cells that were grown on coverslips. Thirty-six hours after transfection COS cells were washed with PBS, fixed with 70% ice-cold methanol, and examined by fluorescence microscopy.Western blotting After stimulation 106 neutrophils/point were lysed in 1 × Laemmli sample buffer. Protein samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore, Bedford, MA). The membranes were probed with antiphospho-CREB-Ser133 antibodies (New England Biolabs) and swine antirabbit peroxidase-conjugated antibodies (DAKO, Glostrup, Denmark), following detection by enhanced chemiluminescence (ECL, Amersham-Pharmacia, Biotech, The Netherlands). Protein kinase expression controls were performed by using 12CA5 and rabbit antimouse peroxidase-conjugated antibodies (DAKO) to detect HA_CKLiK, HA_ERK, and HA_PKB. Detection of CaMKK-VSV was performed with anti-VSV antibodies and swine antirabbit peroxidase-conjugated antibodies. Hybridizations were followed by detection with ECL (Amersham).
Cloning and expression of CKLiK Although several kinases have been implicated in the control of granulocyte effector functions,12,30 most of these proteins are widely expressed. To identify novel granulocyte-specific kinases, degenerate primers against conserved kinase catalytic domains were used as previously described.21 The central core of the catalytic domain consists of subdomains VI and IX and contains 2 well-conserved amino acid triplets APE and DGF.31 Degenerate oligonucleotides were designed against subdomains VI and IX and used in a PCR on cDNA of human PMNs. Under stringent conditions PCR products of approximately 200 bp were amplified, cloned, and sequenced. All cloned PCR products contained the 2 conserved amino acid triplets and many were identical to previously identified kinases (data not shown). However, one clone exhibited homology with calcium/calmodulin-dependent kinase I (CaMKI) and subsequently termed CKLiK (CaMKI-like kinase).To isolate full-length CKLiK cDNA, a human eosinophil library was
screened with the amplified PMN cDNA PCR fragment. Several positive
clones were isolated and sequenced, and one cDNA of 1.7 kb contained a
357 amino acid open reading frame encoding a protein of 40 kd as
determined by in vitro transcription/translation (data not shown). In
Figure 1 the complete cDNA and the
translated amino acid sequence is depicted (accession number
AF286366). The context of the ATG codon was in good agreement
with eukaryotic translation start-sites.32 As shown in
Figure 2A, comparison of the predicted
CKLiK protein sequence in BLAST database revealed a 77% homology of
CKLiK with CaMKI on the amino acid level. The greatest divergence
between the 2 sequences occurs at the N- and C-termini.
To determine the distribution of CKLiK expression we analyzed mRNA levels in primary human leukocytes and hematopoietic cell lines by RNase protection (Figure 2B). The mRNAs from lymphocytes, which include T and B cells, monocytes, and PMNs, were analyzed. Additionally, 3 myeloid cell lines HL60 (promyelocytic leukemia), U937 (histiocytic lymphoma), and THP1 (acute monocytic leukemia) were analyzed for CKLiK expression. As an internal control we used a GAPDH probe (Figure 2B; lower panel). Human PMNs contain high levels of CKLiK (Figure 2B; lane 3), whereas little to no expression was observed in monocytes and lymphocytes (Figure 2B; lanes 1 and 2). In the different lymphoid and myeloid cell lines we could not detect CKLiK (Figure 2B; lanes 4-6). Because CKLiK expression is apparently only detected in mature myeloid cells, we analyzed the expression of CKLiK during differentiation of CD34+ cord blood stem cells toward the neutrophilic lineage. Although after 10 days of differentiation CKLiK mRNA was present, increased mRNA levels are indeed detected during terminal differentiation at day 28 (Figure 2C). Similar results were obtained during the differentiation toward eosinophils in the presence of IL-5 (data not shown). CKLiK kinase activity is dependent on Ca++/calmodulin and can regulate CREM and CREB As mentioned, CKLiK has homology with the Ca++/calmodulin-dependent kinase family. The CaM kinases belong to the serine/threonine class of kinases and include CaMKI, CaMKIV, and the upstream CaMKK.33 To investigate whether Ca++ and calmodulin regulated CKLiK we used CRE-binding protein (CREB) or CRE modulator (CREM) as downstream targets. CREB and CREM belong to the basic-leucine-zipper (bZip) class of transcription factors. Serine 133 for CREB and serine 117 for CREM are phosphorylated by several kinases including CaMKI in vitro,28,34 resulting in interaction with transcriptional coactivators and induction of transcription.35CKLiK kinase activity was analyzed by in vitro kinase assay, using CREM
as a substrate and by transcriptional reporter assays using CREB_GAL4
fusion constructs.28 CKLiK was immunoprecipitated from
transfected COS cell lysates and immunocomplex kinase assays performed
(Figure 3A). CREM
C-terminal truncation of CKLiK results in a constitutively active mutant Sequence alignment with CaMKI and the results described above suggests that CKLiK contains a calmodulin-binding domain regulating its activity. To investigate this hypothesis we generated 2 truncation mutants (Figure 4A). Truncation of the predicted calmodulin-binding domain (CKLiK-309) should result in an inactive kinase. Indeed mutant CKLiK-309, lacking residues 310 to 357, remains inactive even in the presence of Ca++/calmodulin, unable to phosphorylate CREM (Figure 4B) or to activate CREB-mediated transcription after ionomycin stimulation (Figure 4C). However, if CKLiK also contains an autoinhibitory domain similar to CaMKI, then removal of this domain (CKLiK-296) should generate a constitutively active kinase. As was predicted, truncation of residues 297 to 357 generated a constitutively active CKLiK. CREM was phosphorylated by CKLiK-296 in the absence of Ca++/calmodulin (Figure 4B) and CKLiK-296 greatly enhanced CREB-mediated transcription, which was not further enhanced by ionomycin addition (Figure 4C).
Because we demonstrated that activated CKLiK could phosphorylate CREM and activate CREB-mediated transcription, we were interested in determining whether CKLiK was located in the cytoplasm or nucleus. We generated GFP containing fusion constructs of CKLiK-WT and the active CKLiK-296. COS cells were grown on coverslips and transfected with CKLiK and CKLiK-296 containing N-terminal GFP fusion. Localization was examined by fluorescence microscopy (Figure 4D). GFP-CKLiK was clearly located in the cytoplasm, whereas the active form of CKLiK exhibited a strong nuclear fluorescence. Because the active form of CKLiK is detected in the nucleus, it suggests that CKLiK may regulate transcription through nuclear localization. Ca++/calmodulin kinase kinase on the CKLiK activity we performed kinase and
reporter assays. Cotransfection of CaMKK with CKLiK resulted in an
enhanced CREM phosphorylation (Figure 5A;
right panel). In the absence of Ca++/calmodulin in the
kinase assay, CaMKK enhanced CKLiK-induced CREM phosphorylation
(Figure 5A; left panel). Similar results were found using reporter
assays (Figure 5B). Cotransfection of CKLiK with CaMKK resulted in
an elevated reporter activity both in untreated (Figure 5B; left panel)
and ionomycin-treated cells (Figure 5B; right panel). CaMKK alone
had no effect on CAT activity, suggesting that CaMKK cannot itself
activate CREB directly. Interestingly CaMKK can enhance CKLiK
activity in the absence of Ca++ in the in vitro kinase
assay. Indeed there are indications for CaMKK-induced CaMK activity
in the absence of Ca++ and calmodulin33,.39
However, Ca++/calmodulin is present in the transfected
cells, and phosphorylation of CKLiK by CaMKK may lead to
stabilization of the CKLiK/Ca++/calmodulin complex,
resulting in enhanced kinase activity.
Activation of CKLiK by IL-8 in bone marrow-derived myeloid precursor cells Because agonist stimulation of G protein-coupled receptors results in changes of [Ca++]i, these receptors are potential activators of CKLiK. The IL-8 receptor is highly expressed on human neutrophils and IL-8 can activate neutrophil effector functions, such as chemotaxis. To investigate whether IL-8 can activate CKLiK, we used a 32D model system. The 32D cells are IL-3-dependent myeloid precursor cells derived from bone marrow.40 Because these cells express endogenous IL-8R41 we generated stable 32D cell lines expressing HA_CKLiK and used these cells to measure IL-8-induced CKLiK activity. IL-8 was indeed able to induce an increase in intracellular Ca++ in these cells (Figure 6A). IL-8 stimulation of HA_CKLiK-expressing 32D cells resulted in a rapid transient increased CKLiK activation (Figure 6B), whereas in Ca++-depleted cells, IL-8-induced CKLiK activity was completely abolished (data not shown). CKLiK activation parallels the rise in intracellular Ca++ induced by IL-8 (Figure 6A). In addition, W7, an antagonist of calmodulin, completely inhibited the IL-8-induced CKLiK activity (Figure 6C). CKLiK activity was also inhibited by W7 in the ionomycin-treated cells, indicating that calmodulin is indeed critical for the CKLiK kinase activity stimulated by agents, which induce a Ca++ influx.
Activation of downstream signaling pathways by CKLiK To identify potential downstream targets of CKLiK, we analyzed the effect of CKLiK on the activation of the ERK1 MAP kinase and PKB, an effector of PI-3K. The regulation of ERKs and PKB by [Ca++]i has been demonstrated in several systems.42,43 Cells were transfected with HA-tagged ERK or PKB and cotransfected with constitutively active CKLiK (CKLiK-296) or CaMKK and immunocomplex kinase assays were performed. Stimulation
with 20% FCS for 10 minutes was used as a positive control.
Cotransfection of CKLiK-296 had no effect on PKB activity, whereas
cotransfection of CaMKK potently activated PKB similar to levels
induced by 20% FCS (Figure 7A; upper
panel). This is in agreement with the direct PKB phosphorylation by
CaMKK described by Yano and colleagues.42 Interestingly,
however, cotransfection of constitutively active CKLiK-296 resulted in an increase in ERK1 activation (Figure 7A; lower panel). To avoid the
possibility of autocrine effects due to overexpression of active CKLiK,
we constructed a tamoxifen-inducible active CKLiK (ER_CKLiK-296). When
transfected into cells, ER_CKLiK-296 is actively repressed by binding
of heat-shock proteins. On addition of the estrogen derivative,
4-hydroxy-tamoxifen (4-OHT), heat-shock proteins dissociate and
CKLiK-296 is directly activated.44 This system has been
successfully used for other protein kinases.45 Cells were
transfected with ER_CKLiK-296 and HA epitope-tagged ERK1 or PKB.
Before harvesting, cells were stimulated with 4-OHT and ERK/PKB kinase
activity was measured. After 2 minutes of treatment an increased ERK
activation was detected, whereas no PKB activation occurred (Figure
7B). The CKLiK-induced ERK activity could be blocked by the MEK
inhibitor PD098059 (Figure 7C), suggesting that CKLiK activates
signaling molecules upstream in the MAPK pathway. Thus, these results
suggest a role for CKLiK in regulation of ERK MAPK kinases at the level
of MEK or higher.
We have described the identification and characterization of a novel protein serine kinase that is highly expressed in human PMNs. These cells, which include neutrophils and eosinophils, are characterized by a variety of specific effector functions that regulate host defense. Although several protein kinases have been previously postulated to regulate some of these effector functions through pharmacologic inhibition studies, these proteins are ubiquitously expressed and thus more likely to be involved in general cellular processes. Furthermore, the use of pharmacologic inhibitors is often complicated by nonspecific effects15,46 By using degenerate PCR primers against conserved catalytic kinase domains in PCR on cDNA templates from human granulocytes, we have identified a novel protein kinase with an open reading frame of 357 amino acids (Figure 1). This novel kinase shows homology with calcium/calmodulin-dependent kinase I (CaMKI) and therefore we have termed this kinase CaMKI-like kinase (CKLiK). CaMKI belongs to the family of Ca++-and calmodulin-dependent kinases, which includes CaMK I, II, and IV as well as myosin light chain kinase, phosphorylase kinase, and elongation factor 2 kinase.47,48 CaMKI has a wide tissue distribution and can phosphorylate a number of substrates in vitro, including the synaptic vesicle-associated proteins synapsin 1 and synapsin 249 and CREB.28 Interestingly, analysis of CKLiK mRNA in hematopoietic cells revealed high and almost exclusive expression levels in PMNs, suggesting a role for CKLiK in human granulocytes (Figure 2). Up-regulation of CKLiK mRNA was also observed in cord blood CD34+ stem cells differentiated toward neutrophils, which may indicate the importance of this kinase in terminally differentiated myeloid cells. In granulocytes, changes in [Ca++]i have been associated with multiple functions, including degranulation, phagosome-lysosome fusion, regulation of cytoskeletal binding proteins, and transcriptional control.16,17 These are thus potential processes whereby CKLiK may play a role. Based on crystal structure and mutational analysis of CamKI, a model has been proposed for CaMKs regulation.50,51 When Ca++ levels rise within the cytosol, calmodulin binds Ca++ and is capable of interacting with calmodulin-binding proteins such as CaMK. Inactive CaMKs are in a folded configuration whereby the N-terminus acts as a pseudosubstrate by interacting with an autoinhibitory domain. On Ca++ recruitment, calmodulin binds to the calmodulin-binding domain located on the C-terminal of CaMK, which then results in unfolding and autophosphorylation of CaMK. It appears that CKLiK is also similarly regulated because truncation of the predicted calmodulin-binding domain or the autoinhibitory domain resulted in an inactive and constitutively active CKLiK, respectively (Figure 4). Additionally we show that CKLiK can directly activate 2 members of the CREB/ATF family of transcription factors, CREM and CREB, by phosphorylation of serine residues 117 and 133, respectively (Figure 3). Although CKLiK is located in the cytoplasm, the constitutively active mutant was clearly translocated to the nucleus (Figure 4D). Possibly this enhanced nuclear translocation uncouples CKLiK from normal regulation resulting in enhanced transcriptional activation. For CaMKIV it has been suggested that it can phosphorylate CREB in vivo because it shows nuclear localization.52 Although a role for CREB in myeloid cells has been proposed in mediating cytokine or chemokine effector functions,53-56 it remains to be determined if CREB and CREM are physiologic substrates for CKLiK in human granulocytes. Indeed we were able to demonstrate CREB phosphorylation by IL-8 in human PMNs (data not shown). Cotransfection of CaMK kinase (CaMKK The IL-8-induced CKLiK activation was rapid and corresponded to the IL-8-induced rise in [Ca++]i (Figure 6). The IL-8 receptor is highly expressed on human neutrophils and could be a possible upstream physiologic receptor for CKLiK. IL-8 is known to induce chemotaxis, and recently a role for intracellular Ca++ in IL-8-induced migration has been described indicating a possible role of CKLiK in this process.58,59 A role for Ca++ and calmodulin-dependent kinases in apoptosis has also been recently demonstrated because CaMKK was found to directly activate PKB and thereby rescue apoptosis.42 Although we found no activation of PKB by CKLiK, we demonstrated an elevated ERK activity by inducing CKLiK activation, which could be blocked by the MEK inhibitor PD098059 (Figure 7). As previously alluded to, pharmacologic inhibitory studies have suggested that ERK MAP kinases possibly play a role in chemotaxis or respiratory burst and PAF release.6,7,60 The mechanism by which CKLiK can activate ERKs however remains to be resolved, although it presumably occurs upstream of ERK. In conclusion, the approach of degenerate PCR resulted in a successful isolation of a novel PMN protein kinase. We have demonstrated that this kinase is highly and predominantly expressed in human PMNs and terminally differentiated myeloid stem cells and that Ca++/calmodulin regulates its activity. IL-8, which is a potent activator of neutrophil effector functions, is capable of activating CKLiK in bone marrow-derived myeloid cells. Furthermore, a role for CKLiK is suggested in regulation of ERK MAP kinases because induced CKLiK activity was sufficient to activate ERK1. Therefore, the restricted expression of CKLiK in granulocytes can allow the transduction of granulocyte-specific signals induced by a general signal transduction event: the rise in [Ca++]i, initiated by inflammatory mediators.
We would like to thank Rolf de Groot for supplying the CREM
Submitted April 18, 2000; accepted July 6, 2000.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Paul J. Coffer, Department of Pulmonary Diseases, G03550, University Medical Centre Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands; e-mail: P.Coffer{at}hli.azu.nl.
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  Z. He, J. Jiang, M. Kokkinaki, N. Golestaneh, M.-C. Hofmann, and M. Dym Gdnf Upregulates c-Fos Transcription via the Ras/Erk1/2 Pathway to Promote Mouse Spermatogonial Stem Cell Proliferation Stem Cells, January 1, 2008; 26(1): 266 - 278. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
ZAPII eosinophilic
library
).The amino acid sequence is indicated on the right. (B)
Distribution of CKLiK was determined by RNase protection. The PCR
product encoding the catalytic domain of CKLiK was used to generate a
radiolabeled RNA probe. RNA isolated from primary hematopoietic cells
and cell lines were hybridized for CKLiK expression. A GAPDH probe was
used as a control. Lanes 1 to 3 represent primary leukocytes (1, lymphocytes; 2, monocytes; 3, PMNs) and lanes 4 to 6 represent lymphoid
and myeloid cell lines as indicated. (C) Expression of CKLiK in cord
blood-derived CD34+ stem cells during differentiation
toward neutrophils was analyzed by RNase protection as in panel B. Time
points indicate the number of days differentiated toward neutrophilic
lineage in the presence of G-CSF.
) or without (
) 1 µmol/L ionomycin
and reporter assays were performed. Data represent at least 3 independent experiments ± SEM and were corrected for transfection
efficiency. (D) Active CKLiK is localized in the nucleus. Cells were
grown on coverslips and transfected with CKLiK and active CKLiK-296
containing N-terminal enhanced green fluorescent protein (eGFP).
Thirty-six hours after transfection cells were examined by fluorescence
microscopy for CKLiK localization.
