Casein kinase I delta (CKIδ) is involved in lymphocyte physiology

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European Journal of Cell Biology 82, 369 ± 378 (2003, July) ¥ ¹ Urban & Fischer Verlag ¥ Jena http://www.urbanfischer.de/journals/ejcb

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Casein kinase I delta (CKId) is involved in lymphocyte physiology Tanja Maritzen2), J¸rgen Lˆhler, Wolfgang Deppert, Uwe Knippschild1) Heinrich-Pette-Institut f¸r Experimentelle Virologie und Immunologie an der Universit‰t Hamburg, Hamburg/Germany Received November 14, 2002 Received in revised version February 3, 2003 Accepted February 26, 2003

Casein kinase I d ± lymphocytes ± proliferation ± differentiation ± lymphoma

Introduction

The casein kinase I isoform delta (CKId) plays an important role in vesicular trafficking, chromosome segregation, cell cycle progression, cytokinesis, developmental processes, and circadian rhythm. In this study we examined the distribution pattern of CKId and quantified its kinase activity in various tissues of BALB/c mice. Whereas CKId is ubiquitously expressed, differences in the kinase activity were detected in organs with comparable CKId protein levels. To elucidate the role of CKId in splenocytes, which displayed the highest kinase activity, the cell type-specific distribution of CKId within the spleen was investigated. Immunohistochemical analysis revealed a strong CKId immunolabeling in lymphoid cells of the white pulp, while in the red pulp CKId immunoreactivity was found in cells of various haematopoietic lineages. Furthermore, high CKId kinase acitivity was observed in isolated lymphocytes and granulocytes of young BALB/c mice. In lymphocytes the CKId activity increased upon mitogenic stimulation, whereas upon girradiation CKId protein and activity levels were diminished. Interestingly, the comparison of CKId activity in p53‡/‡ and p53 / lymphocytes revealed a higher activity in p53‡/‡ lymphocytes. In addition, we observed an increased immunostaining in cells of hyperplastic B follicles and advanced B-cell lymphomas in p53-deficient mice. Thus, our results indicate that CKId plays several roles in lymphocyte physiology.

CKId is a member of the casein kinase I family of serine/ threonine protein kinases that is ubiquitously expressed in eukaryotic cells. So far, several CKI isoforms, namely a, b, g, d, and e (Gross and Anderson, 1998; Fish et al., 1995; Graves et al., 1993; Rowles et al., 1991) and their various splice variants (Zhai et al., 1995) have been characterized in mammals. Although differing in tissue distribution and subcellular localisation, all these isoforms share several functional and structural characteristics. They exclusively use ATP as phosphate donor, are generally co-factor-independent and need N-terminal acidic and/or phosphorylated amino acids for substrate recognition. All CKI isoforms exhibit a high degree of sequence conservation within their kinase domains, but differ significantly in length and primary structure of their N-terminal and Cterminal non-catalytic domains. The variable C-terminal domains are responsible for the substrate specificity of the different isoforms and are involved in the regulation of the kinase activity (Cegielska et al., 1998; Graves and Roach, 1995; Gietzen and Virshup, 1999). The casein kinases I are able to phosphorylate a wide spectrum of substrates suggesting an involvement in different cellular processes. While the participation of the yeast CKI homologues in the regulation of membrane transport (Murakami et al., 1999; Panek et al., 1997), cell morphogenetic processes (Robinson et al., 1993) and DNA repair pathways (Hoekstra et al., 1994) is well established, the role of the mammalian CKI isoforms has still to be elucidated in detail. CKId as well as CKIe have been identified as key regulators of developmental processes due to their ability to influence the Wnt pathway (reviewed in (McKay et al., 2001; Vielhaber and Virshup, 2001)) and as important participants in the regulation of the circadian rhythm (reviewed in (Whitmore et al., 2000)) (Camacho et al., 2001; Lowrey et al., 2000). There is also increasing evidence for involvement of CKId in Alzheimer disease (Kuret et al., 1997; Walter et al., 1998; Yasojima et al., 2000; Schwab et al., 2000). Furthermore, CKId as well as several other casein kinase I isoforms have been implicated in regulating apoptotic processes in certain cell types (Desagher

Abbreviations. ConA Concanavalin A. ± LPS Lipopolysaccharide. ± PALS Periarteriolar lymphatic sheath.

1)

Dr. Uwe Knippschild, Chirurgische Universit‰tsklinik Ulm, Steinhˆvelstr. 9, D-89075 Ulm/Germany, e-mail: [email protected], Fax ‡ 49 731 500 209. 2) Present address: Zentrum f¸r Molekulare Neurobiologie Hamburg (ZMNH), Falkenried 94, D-20251 Hamburg/Germany.

0171-9335/03/82/07-369 $15.00/0

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et al., 2001). The induction of CKId by the tumor suppressor p53 in stress situations and its ability to phosphorylate p53 suggests a further role for CKId in modulating the effects of p53 on cell growth and genome integrity (Knippschild et al., 1996, 1997; Behrend et al., 2000a). Additionally, the phosphorylation of tubulin and microtubule-associated proteins points to a function of CKId in the regulation of microtubule dynamics and chromosome segregation (Behrend et al., 2000b). In order to obtain detailed information about the physiological functions of CKId in vivo, we determined the tissuespecific distribution of CKId in young adult mice. Our ongoing work on the tissue-specific distribution of CKId revealed a high CKId protein level and activity in the spleen. Immunohistochemistry indicated that lymphoid cells within the white pulp as well as cells of different haematopoietic lineages within the red pulp show a strong CKId immunostaining. Since CKId protein and activity levels in lymphocytes increase after mitogenic stimulation, a participation of CKId in the regulation of lymphocyte proliferation and antigen-dependent differentiation seems likely. In contrast, g-irradiation, which causes the induction of p53-dependent apoptotic processes in lymphocytes, leads to a dramatic decrease in CKId activity and protein levels. This observation suggests that CKId might be an important regulator of p53 activity. Deregulation of both, the p53 pathway and CKId activity, may also synergize in neoplastic processes. Indeed, we observed an increased immunostaining in cells of hyperplastic B follicles and advanced B-cell lymphomas in p53-deficient mice. Taken together, our observations point to an important role of CKId in lymphocyte physiology.

Materials and methods Antibodies The peptide CFRIPASQTSVTFDHLG (human CKIe-specific C-terminal sequence) was coupled to keyhole limpet hemocyanin for the first, to bovine serum albumin for the second, and to ovalbumin for the third immunisation. For the immunisation of rabbits the coupled peptide, emulsified in an equal volume of complete (first immunisation) or incomplete (subsequent immunisations) Freud×s adjuvant (Gibco Laboratories, Grand Island, NY, USA), was used for subsequent subcutaneous injections. A test bleed of each animal was taken 10 days after each boost, and after 3 to 4 boosts the animals were bled out. The specificity of the polyclonal antiserum (712) was confirmed by Western blot analysis using the bacterially expressed GST-CKIe fusion protein (FP455) (Knippschild et al., 1997) as substrate. Additionally, FITC-labeled anti-CD3e and PE-labeled anti-CD45R/ B220 antibodies were purchased from BD PharMingen (Franklin Lakes, NY, USA). The polyclonal rabbit anti-CKId antiserum (NC10) is described in (Behrend et al., 2000b) and the CKId-specific monoclonal antibody 128A was obtained from ICOS (Bothell, Washington, USA).

Animals and tissue processing Four-week-old BALB/c mice were killed, and tissues and organs, including muscle, lung, salivary glands, pancreas, thymus, duodenum, heart, brain, liver, spleen, eye, ovary, epididymis, and testis were harvested, homogenized and lysed in 50 mM Tris-HCl (pH 8.0) containing 120 mM NaCl, 10% glycerol, 0.5% NP40, 5 mM EDTA, 1 mM EGTA, 5 mM dithiothreitol, 1 mM benzamidine, 4 mg/ml leupeptin, and 30 mM aprotinin when used for Western Blot analysis. Alternatively, homogenized tissues and isolated lymphocytes and granulocytes were lysed in 20 mM Tris-acetate (pH 7.0), 0.27 M sucrose, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM benzamidine, 4 mg/ml leupeptin, 30 mM aprotinin, and 0.1% b-mercaptoethanol. Cell lysates were passed through 0.2-mm filters, and equal amounts of protein (4 mg/ml) were

applied to a Uno Q (BIO-RAD, Munich, Germany) or to a Mini Q column (Amersham, Freiburg, Germany) attached to an AEKTA purifier (Amersham). The proteins were eluted with a linear gradient of sodium chloride in buffer B (50 mM Tris-HCl (pH 7.5), 1 M NaCl, 1 mM EDTA, 1 mM EGTA, 5% glycerol, 0.03% Brij35, 1 mM benzamidine, 0.1% b-mercaptoethanol).

Immunohistochemistry Fixed tissue specimens from BALB/c wt and BALB/c p53 / mice were obtained either by perfusion fixation with 4% formalin containing 1% acetic acid in deep anesthesia via the circulation or by immersion fixation of the organs with the same fixative. Deparaffinated sections were stained with hematoxylin and eosin according to standard laboratory procedures. For CKId immunostaining on paraffin sections, an indirect immunoperoxidase method was used. Briefly, for antigen retrieval, deparaffinated sections were treated with a commercial ™Target Unmasking Fluid∫ (TUF, DAKO Diagnostika) at 98 8C in a microwave oven. Incubation with 1 : 1200 diluted primary polyclonal rabbit anti-CKId antiserum (NC 10) (Behrend et al., 2000b) was done overnight at 4 8C. Specifically bound antibodies were detected using a highly sensitive peroxidase- and polymer-conjugated goat anti-rabbit Ig detection system (Envision, DAKO Diagnostika). Peroxidase activity was revealed by the diaminobenzidine technique (DAB plus Kit, DAKO Diagnostika, Hamburg, Germany). Finally, sections were counterstained with hemalum and permanently coverslipped.

Cell preparation, cultivation, stimulation and irradiation BALB/c wt and BALB/c p53 / mice (Donehower et al., 1992), 4 to 6 weeks old, were killed by cervical dislocation, and their spleens were disrupted by passing through a stainless steel sieve yielding single cell suspensions. Lymphocytes were purified on a Ficoll gradient as described by Boyum (1968). The pellet, containing erythrocytes and granulocytes, was washed with phosphate-buffered saline (PBS). Erythrocytes were then lysed, and the remaining granulocytes were washed twice with PBS. Enrichment of lymphocytes was demonstrated by FACS analysis using FITC-labeled anti-CD3e or PE-labeled antiCD45R/B220 antibodies. The proportion of T and B cells increased; the B cell population rose from 24% to 47% of the total cell population. Isolated lymphocytes and granulocytes were cultured in RPMI-1640 medium with 5% fetal calf serum (FCS) and 50 mM b-mercaptoethanol, 100 U/ml penicillin and 100 mg/ml streptomycin at a density of 1  106 cells/ml. Untreated controls and cells treated with concanavalin A (ConA, 3 mg/ml, Sigma, Germany), ConA and the CKId/e-specific inhibitor 3-[(2,4,6-trimethoxyphenyl)methylidenyl]-indolin-2-one (IC261) (ConA, 3 mg/ml; IC261, 1 mM) or with lipopolysaccharide (LPS, 75 mg/ml, Sigma) were cultured at 37 8C in a 5% carbon dioxide incubator. g-Irradiation of lymphocytes was performed 30 min after isolation or 30 min after stimulation using a dosis of 5 Gray.

Assay of lymphocyte DNA synthesis Lymphocytes enriched by Ficoll gradient purification were cultured in 0.18 ml of RPMI-1640 at a concentration of 4  106 ml 1 (7.2  105 cells per well) in flat-bottom plastic microtiter plates. They were cultured in the presence or absence of lectin (3 mg/ml ConA) for a total of 54 h at 37 8C in a 5% carbon dioxide incubator. Where indicated, 1 mM IC261 was added 4 h after cell stimulation with ConA. At the time points indicated cells were labeled with 5 mCi of [3H]thymidine. One hour later cells were harvested using a multiple cell harvester (Skatron), and dried filters were counted in a liquid scintillation counter. In these experiments triplicate cultures were measured for each time point.

Expression and purification of glutathione Stransferase (GST) fusion proteins The GST fusion proteins FP267 (encoding the first 64 amino acids of wild-type mouse p53 with potential phosphorylation sites for CKI (Milne et al., 1995)) and FP454 (encoding human CKIe (Knippschild et al., 1997)) were isolated by incubating the bacterial lysates with

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Involvement of CKId in lymphocyte physiology 371

gluthatione-Sepharose 4B beads (Amersham) at 4 8C for 1 h. The beads were washed three times with ice-cold PBS and once with 50 mM TrisHCl (pH 7.5). Fusion proteins were eluted from the beads by incubation with 50 mM Tris-HCl buffer (pH 7.5), containing 10% glycerol and 5 mM reduced glutathione. Eluted fusion proteins were dialyzed against 50 mM Tris-HCl (pH 7.5), containing 10% glycerol and stored at 70 8C.

Detection of CKId kinase activity in splenic cells

Tissue extracts, isolated lymphocytes and granulocytes of p53‡/‡ and p53 / BALB/c mice were prepared as described, and equal amounts of protein were applied to anion exchange columns attached to an AEKTA purifier. The proteins were eluted with a linear gradient of sodium chloride and each fraction was screened for CKId activity using the purified GST-mouse p531±64 fusion protein FP267 as substrate. FP267 was resuspended in a total volume of 20 ml containing 25 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 0.1 mM EDTA, and 20 mM [g-32P]ATP. The reactions were initiated by addition of enzyme (usually 2 ml of 0.5 ml fractions eluted from the Mini Q column) and incubated at 30 8C for 30 min. Where indicated, in vitro kinase assays were performed in the presence of IC261, which was synthesized as described elsewhere (Mashhoon et al., 2000). Reaction products were separated on 12.5% SDS gels and the phosphorylated proteins were visualized by autoradiography. For quantitative analysis, the phosphorylated products were excised from the gel, and Cerenkov radiation was measured using a scintillation counter.

Western blotting Steady-state levels of CKId were determined by Western blot analysis. Tissue and cell lysates were separated by SDS-PAGE, proteins were transferred to an Immobilon-P membrane (Millipore, Hamburg, Germany) and probed with the CKId-specific monoclonal antibody 128A (ICOS), the CKIe-specific polyclonal rabbit serum 712 or with an actin-specific monoclonal antibody (Chemicon). Detection was carried out using horseradish peroxidase-conjugated anti-mouse IgG or antigoat IgG as secondary antibodies, followed by chemiluminescence detection (ECL, Amersham).

Results CKId protein levels in organs of young adult BALB/c mice To characterize the CKId expression levels in various organs of BALB/c mice, equal protein amounts of the different tissue extracts were separated by SDS-PAGE and analyzed by Western blotting. CKId was ubiquitously detectable (Fig. 1A), but the level of CKId immunoreactivity differed considerably between the organs. Whereas CKId protein levels were high in testis, epididymis, ovary, brain, thymus, pancreas, and the lungs, intermediate CKId protein levels were found in the liver and duodenum. Only low protein amounts were detected in the salivary glands, the eye and the heart. In some cases hypershifted forms of CKId possibly due to posttranslational modifications, were present in the spleen, ovary, thymus, and lungs. In tissues with high proteolytic activity, such as the salivary glands, increased amounts of degraded forms of CKId could be observed (Fig. 1A).

CKId kinase activity in organs of young adult BALB/c mice To investigate the CKId kinase activity in the positively tested tissue specimens, cell extracts were prepared from brain, testis, spleen and thymus, all of which had shown comparable CKId protein levels by Western blotting. Each extract was fractionated by ion exchange chromatography using a Uno Q column (BIO-RAD), and the eluted fractions were tested for kinase

Fig. 1. CKId expression in organs of 4-week-old BALB/c mice. A. Comparison of CKId protein levels. Specimens were homogenized and equal protein amounts (50 mg) were separated by SDS-PAGE. CKId protein levels were detected by Western blotting using the CKIdspecific monoclonal antibody 128A (ICOS). B. CKId kinase activity. Tissue samples from spleen, brain, testis, and thymus were lysed and soluble extracts were prepared as described in Materials and methods. Equal protein amounts of each extract (6 mg) were loaded onto a 1-ml Uno Q column (BIO-RAD) and eluted with a linear gradient of increasing NaCl concentration (solid diagonal line). Fractions (0.5 ml) were collected, and kinase activity was determined. Kinase activity is represented by closed squares (spleen), open squares (brain), closed circles (testis), and open circles (thymus). C. Detection of CKId by Western blot analysis. Western blot analysis of the kinase peak fractions from different organs reveals the presence of 2 to 3 CKId-specific signals.

activity using the CKId/e-specific GST-p531±64 fusion protein FP267 as a substrate. In each case the kinase activity eluted at about 130 mM NaCl; it was maximal in the splenic peak fraction (Fig. 1B). In the brain and the testis, we observed a moderate CKId activity, whereas CKId activity was very low in the thymus. CKId specificity of this kinase activity was confirmed by two different approaches. First, fractions containing kinase

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activity were tested for the presence of CKId by Western blot analysis using the CKId-specific monoclonal antibody 128A (Fig. 1C). In contrast to CKId, CKIe could never be detected in Western blot analysis using the CKIe-specific antibody 712 (data not shown). Second, in vitro kinase assays were performed in the absence and in the presence of the CKId/especific inhibitor IC261 (Mashhoon et al., 2000) using FP267 as a substrate and the splenic kinase peak fraction 8 as enzyme. In the presence of the CKId/e-specific inhibitor IC261 (1 mM), an 80% reduction of kinase activity was observed compared to control reactions (data not shown), pointing to CKId as the source of the kinase activity present in the peak fractions.

Protein levels and enzymatic activity of CKId in splenic cells Having recognized a very high CKId activity in the spleen, our further studies focused on determining the role of CKId in regulating cellular functions of splenic cells. Anti-CKId immunostaining of splenic tissue sections revealed an intense labeling of a portion of lymphoid cells within the white pulp. These cells were predominantly localized in the periarteriolar lymphatic sheath (PALS) and in the marginal zone, while lymphoid cells of the B cell follicles displayed a relatively weak CKId immunostaining signal (Fig. 2A). In the red pulp, CKId staining was detected in cells of different haematopoietic lineages (Fig. 2B). In typical perisinusoidal, paratrabecular and subcapsular locations, plasma cells (Fig. 2B, 4) exhibited a strong cytoplasmic CKId immunostaining. CKId immunostaining was also found to be especially strong in immature granulocytic cells (Fig. 2B, 1) located close to the trabecules and the capsule. Erythroid cells and megakaryocytes (Fig. 2B, 3) exhibited a weaker cytoplasmic CKId immunostaining. To determine whether the high CKId protein levels in lymphocytes and granulocytes were associated with a corresponding kinase activity, lymphocytes and granulocytes were isolated from spleen cell suspensions using a Ficoll gradient as described in Materials and methods. Lysates of these cells were fractionated by ion exchange chromatography. Each fraction was then tested for kinase activity using the CKId/e-specific GST-p531±64 fusion protein FP267 as substrate. In the peak fractions of lysates from both cell types similar kinase activity levels were detected that eluted at a concentration of 130 mM NaCl (Fig. 3).

CKId protein levels and kinase activity of activated lymphocytes In order to evaluate the physiological function of CKId in splenic lymphocytes we first examined the role of CKId during lymphocyte proliferation. For this purpose, isolated lymphocytes of young adult BALB/c mice (4 weeks old) were activated with the T cell mitogen ConA. Cell extracts from ConAstimulated and unstimulated lymphocytes taken at different time points were tested for CKId protein levels by Western blotting. An increase in the CKId protein level was first detectable 4 h after ConA stimulation, reaching its maximum at 24 h (Fig. 4A). Even 36 h after ConA stimulation, CKId protein levels were still elevated compared to untreated control cells (Fig. 4A). To determine whether this increased CKId protein level is associated with an increase in CKId kinase activity, stimulated as well as untreated lymphocytes were lysed after 17 h of incubation, and equal protein amounts were fractionated by ion exchange chromatography using a linear NaCl

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gradient. The results of the in vitro kinase assays (Fig. 4B) show a significant increase in kinase activity upon ConA stimulation. To determine whether CKId protein level and enzymatic activity was likewise altered in B cells, isolated lymphocytes were treated with the B cell mitogen LPS (Fig. 4A). As ConA in T cells, LPS treatment strongly increased CKId protein levels in B cells, however, already starting at 1.5 h after mitogenic activation. The maximal CKId protein level was reached 16 h after stimulation. Seventy-two hours after stimulation the CKId protein level decreased to that of untreated control cells (Fig. 4A). In summary, both ConA and LPS stimulation led to an increase in CKId protein levels. Our results indicate that the kinetics of the increase in CKId protein level in B lymphocytes upon LPS stimulation differs from that in T lymphocytes after ConA stimulation. Whereas the CKId levels start to increase 4 h after ConA stimulation, this increase is already seen 1.5 h after LPS stimulation. To further analyze the role of CKId in lymphocyte proliferation, we tested the effect of the CKId/e-specific inhibitor IC261 (Mashhoon et al., 2000) on the proliferation rate of stimulated lymphocytes. Isolated lymphocytes were activated with ConA in the absence and presence of IC261. DNA synthesis rates were determined by measuring [3H]thymidine incorporation into the cellular DNA (Fig. 5). While ConA-stimulated lymphocytes showed a maximal DNA synthesis rate 28 h after stimulation, ConA/IC261-treated cells exhibited only a low background of residual DNA synthesis similar to that observed in unstimulated lymphocytes. Similar results were obtained when lymphocytes were activated by LPS (data not shown). Therefore, CKId activity seems to be important for mitogenic activation of lymphocytes.

CKId protein level and kinase activity of girradiated lymphocytes It is known that treatment of fibroblasts and epithelial cells with DNA-damaging agents (e.g. etoposide) or g-irradiation results in a p53-mediated growth arrest associated with a p53dependent upregulation of CKId protein levels and kinase activity (Knippschild et al., 1996, 1997). In contrast, lymphocytes respond to g-irradiation preferentially by the induction of apoptosis. To determine the potential involvement of CKId in response to g-irradiation, unstimulated and ConA-stimulated lymphocytes were treated with a dose of 5 Gray, and CKId protein levels and kinase activity were determined. Lysates of these cells were analyzed by Western blotting using the CKId-specific antibody 128A. Unstimulated as well as ConA-stimulated lymphocytes showed a sharp decline in CKId protein level after g-irradiation. In vitro kinase assays of chromatographically fractionated lysates of unstimulated and ConA-stimulated irradiated cells revealed an 85% decrease in kinase activity 17 h after g-irradiation (Fig. 6A, B). At this time point 77% of girradiated lymphocytes versus 27% of untreated control cells were found to be apoptotic as determined by flow cytometry (data not shown).

CKId protein levels and kinase activity of p53deficient lymphocytes We previously reported that CKId and p53 are regulated interdependently in cell lines of different origins (Knippschild et al., 1997). Therefore, we compared the CKId activity of freshly isolated p53-expressing lymphocytes from young adult mice (4 weeks old) with that of p53-deficient lymphocytes from

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Fig. 2. CKId immunostaining in the spleen of p53 wild-type 4-weekold BALB/c mice. A. White pulp. CKId-positive lymphoid cells are localized in the PALS (P) and within the marginal zone (M). B. Red

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pulp. CKId immunoreactivity in elements of the granulocytic, erythroid and megakaryocytic lineages. Granulocytic cells (1), erythroid cells (2), megacaryocytes (3), plasma cells (4).

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Fig. 3. CKId kinase activity in splenic lymphocytes and granulocytes. Equal amounts of particle-free lysates of lymphocytes and granulocytes (1 mg) were fractionated on a Uno Q column (BIO-RAD). Proteins were eluted with a linear gradient of increasing NaCl concentration (solid diagonal line). The kinase activity was determined in each fraction and is presented as open squares (lymphocytes) and closed squares (granulocytes).

young adult p53 / mice that do not yet show alterations of lymphatic tissues. Lysates of p53 wild-type (p53‡/‡) and p53deficient (p53 / ) lymphocytes were prepared and fractionated. In vitro kinase assays of these fractions revealed that in p53deficient lymphocytes the CKId activity was reduced to about 40% of the activity measured in lymphocytes containing wt p53 (Fig. 7).

Immunohistochemical detection of CKId in hyperplastic B cell follicles and in advanced B cell tumors Among the spontaneously developing multiple tumors of p53deficient mice throughout their lifespan lymphomas are frequently encountered. Since tumor cells are in a proliferative state comparable to that of ConA- or LPS-stimulated cells, it is likely that CKId expression might be upregulated in neoplastically transformed lymphocytes. To test this hypothesis we determined CKId immunolabeling in spleen sections of p53deficient mice. In the white pulp of 6-week-old p53-deficient mice (Fig. 8A) the overall CKId immunostaining in lymphoid cells appeared to be decreased compared to p53 wild-type spleen tissue (Fig. 2A, B). This observation correlates well with our results obtained by in vitro kinase assays (Fig. 7). However, in hyperplastic B cell follicles (Fig. 8A), CKId-positive lymphoblastoid cells exhibit a much more intense CKId immunostaining than the nontransformed B cells. Three months later, after manifestation of advanced B cell lymphoma, tumor cells displayed an intermediate CKId immunoreactivity in their cytoplasm (Fig. 8B).

Discussion Here we report for the first time that the stress-inducible casein kinase I isoform delta (CKId) seems to be involved in several aspects of lymphocyte physiology. Our immunohistochemical analysis revealed that beside plasma cells, erythroid, granulocytic and megakaryocytic cells of the red pulp as well as a portion of lymphocytes of the white pulp exhibit an especially strong CKId immunoreactivity. Interestingly, labeled lymphocytes were recognized in different anatomical compartments of

Fig. 4. CKId protein level and kinase activity in mitogen-stimulated and unstimulated lymphocytes. A. Detection of CKId in resting, ConAor LPS-stimulated lymphocytes by Western blotting. At the indicated time points cells were lysed, and CKId protein levels were detected by Western blotting using the CKId-specific monoclonal antibody 128A (ICOS). ConA as well as LPS stimulation led to an increase in CKId protein levels. In addition, the same samples were probed for equal loading using an actin-specific monoclonal antibody (Chemicon). B. Detection of kinase activity in resting and ConA-stimulated lymphocytes. Equal amounts of each cell extract were loaded onto a 1-ml Uno Q column (BIO-RAD), and the proteins were eluted with a linear gradient of increasing NaCl concentration (solid diagonal line). Fractions (0.5 ml) were collected, and the kinase activity was detected as described in Materials and methods. Open squares indicate the kinase activity in eluted fractions of ConA-stimulated lymphocytes, closed squares that in eluted fractions of resting lymphocytes.

the white pulp, e.g. in the PALS and the marginal zone, where quite diverse lymphocyte subpopulations are encountered such as T cells and B cells, respectively. Thus we suggest that the lymphocytic CKId immunoreactivity is rather reflecting a certain state of cellular activity such as motility or proliferation than a cell population-specific determinant. Therefore, our further studies were aimed to clarify the involvement of CKId in the regulation of elementary functions of T- and Blymphocytes such as proliferative and apoptotic processes.

CKId in activated lymphocytes We were able to show that both, the activation of T and B cells with the polyclonal T cell mitogen ConA and with the B cell mitogen LPS, respectively, caused an increase in CKId expression. The kinetics of the CKId increase upon ConA stimulation differ significantly from that of LPS stimulation. Whereas CKId

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Fig. 7. CKId activity in p53 wild-type and p53-deficient mice. CKId kinase activity was detected in eluted fractions of p53‡/‡ lymphocytes (open squares) and p53 / lymphocytes (closed squares). The result indicates a decrease in CKId activity in p53-deficient lymphocytes. Fig. 5. DNA synthesis in mitogen-stimulated and unstimulated lymphocytes. Lymphocytes were isolated using a Ficoll gradient. Cells (7.2  105), untreated or treated with either ConA or with ConA and IC261, were cultured and labeled with 5 mCi [3H]thymidine at the indicated time points. The amounts of [3H]thymidine incorporation into the cellular DNA are presented as columns (black: control cells; white: ConA-treated cells; grey: ConA/IC261-treated cells).

levels started to rise 4 h after ConA stimulation, increased CKId protein levels were already detected 1.5 h after LPS stimulation. Although protein amounts have not been equalized between both experiments, the capacity of LPS to induce CKId protein expression is probably higher than that of ConA. This assumption is supported by the observation that only a part of Ficoll-isolated lymphocytes has preserved the capacity to be sensitized by ConA (Mosner and Deppert, 1992). However, mitogen-induced activation of lymphocytes could be reversed by the CKId/e-specific inhibitor IC261. Thus, CKId seems to be involved in activation processes of both cell types. However, there might exist several mechanisms by which CKId could contribute to lymphocyte activation. One of several alternatives might be the CKId-specific phosphorylation of transcription factors. Recently, it has been reported that the phosphorylation of the ™Nuclear factor of activated T cells∫ (NFAT) (Marin et al., 2002) by CKIa results in its retention in the cytoplasm (Zhu et al., 1998; Porter et al., 2000). The early cellular responses initiated by lymphocyte stimulation also include the reorganisation of cytoskeletal elements. As CKId has been shown to interact with cytoskeletal proteins such as tubulin (Behrend et al., 2000a), it might be also possible that CKId is involved in cytoskeletal reorganisation processes. Finally, the granular cytoplasmic immunostaining of CKIdpositive lymphocytes could indicate its association with small vesicles. Since CKId has been reported to be involved in vesicle transport along microtubules (Behrend et al., 2000a), this could also hold true for enhanced vesicle trafficking processes in activated lymphocytes.

Role of CKId in g-irradiation-induced apoptosis of lymphocytes and its role in the tumorigenesis of lymphatic tissues of p53deficient mice Fig. 6. CKId protein levels and kinase activity in g-irradiated lymphocytes with and without ConA stimulation. A. Western Blot analysis of CKId. Cells (2  107) were treated as indicated and lysed at the given time points. CKId protein levels were detected by Western blot analysis using the CKId-specific monoclonal antibody 128A (ICOS). Both, resting and stimulated, g-irradiated lymphocytes showed a decrease in CKId protein levels. B. Assessment of CKId activity. Kinase activity was detected in eluted fractions of control cells, of ConA-stimulated (17 h after stimulation) and of g-irradiated lymphocytes with and without ConA stimulation. While CKId activity increased upon ConA treatment (open squares), CKId activity decreased in g-irradiated resting (closed circles) and ConA-stimulated lymphocytes (open circles) compared to control cells (closed squares).

The strong upregulation of CKId in lymphocytes upon mitogenic stimulation is in accordance with the existence of a positive feedback loop between CKId and p53, which has been postulated for epithelial cells and fibroblasts (Knippschild et al., 1997). However, lymphocytes show a decrease in CKId protein levels upon g-irradiation. This obvious discrepancy could be explained by differences in the physiological behavior of lymphocytes compared to that of fibroblasts and epithelial cells. While the applied g-irradiation dose leads to a p53dependent cell cycle arrest in epithelial cells and fibroblasts, the same dose causes a p53-mediated apoptosis in lymphocytes. It has been postulated that p53 functions are necessary for both,

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Fig. 8. CKId immunostaining in the spleen of p53-deficient BALB/c mice. A. Spleen of a 6-week-old p53-deficient mouse. Strong CKIdspecific immunostaining of lymphoblastoid cells of the B cell zone (F).

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B. Lymphoblastic tumor cells of an advanced B cell lymphoma with granular cytoplasmic CKId immunoreactivity. Part of the spleen of a 4month-old p53-deficient mouse.

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mitogen-stimulated activation of lymphocytes and the induction of apoptosis. In each case, p53 functions are probably regulated by changes in the phosphorylation status of individual phosphorylation sites of p53. Differential phosphorylation of the CKId-specific phosphorylation sites of p53 seems to be important to modulate p53 function dependent on the physiological conditions. Whereas upregulation of CKId during mitogenic stimulation increases the phosphorylation of the CKId-targeted p53 phosphorylation sites, downregulation of those sites upon g-irradiation in lymphocytes might affect p53mediated apoptotic functions. Downregulation of CKId has been observed in both p53‡/‡ and p53 / lymphocytes, but only p53-positive lymphocytes undergo apoptosis, illustrating the tight connection between p53 and CKId. Furthermore, the p53independent downregulation of CKId upon g-irradiation points to the existence of additional mechanisms regulating CKId activity; in nerve cells for example the glutamate receptor has been identified as an upstream regulator of CKId (Liu et al., 2001). This assumption is supported by the fact that under physiological conditions only a 40% reduction of CKId kinase activity in the spleen of young p53-deficient mice is observed compared to the activity levels in p53 wild-type mice. In p53-deficient mice the loss of p53 causes the development of tumors, especially lymphomas, at an early age. In this study we observed an increased CKId immunostaining in cells of hyperplastic B follicles and advanced B-cell lymphomas. The proliferative status of tumorigenic B cells might be similar to the status of ConA-stimulated cells, and therefore elevated CKId levels might just be a consequence of the newly acquired proliferative capacity of tumorigenic B cells. Alternatively, upregulation of CKId might be a common feature during cellular transformation. To elucidate the role of CKId during tumorigenesis, additional experiments are necessary. Acknowledgements. This work was supported by a grant from the Deutsche Krebshilfe, Dr. Mildred Scheel Stiftung, to Uwe Knippschild (10 ± 1683-KN2). The Heinrich-Pette-Institut is financially supported by Freie Hansestadt Hamburg and Bundesministerium f¸r Gesundheit. We would like to thank ICOS corporation (Washington, USA) for providing us with the CKId-specific monoclonal antibody 128A and the CKId/e-specific inhibitor IC261, David Meek for providing the GST-Nterminal p53 fusion protein FP267, and Jochen Heukeshoven for peptide synthesis. We thank Nadine Diersch and Karin Heigl for their technical assistance and Ella Kim, Cagatay Gunes, Sonja Wolff, Martin Stˆter, and Peter W¸rl for critical reading of the manuscript and helpful discussions.

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