©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Activation of Ca/Calmodulin-dependent Protein Kinase (CaM-kinase) IV by CaM-kinase Kinase in Jurkat T Lymphocytes (*)

(Received for publication, August 3, 1995; and in revised form, October 1, 1995)

In-Kyung Park Thomas R. Soderling (§)

From the Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201-3098

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Ca/calmodulin-dependent protein kinase IV (CaM-kinase IV), a member of the CaM-kinase family involved in transcriptional regulation, is stimulated by Ca/CaM but also requires phosphorylation by a CaM-kinase kinase for full activation. In this study we investigated the physiological role of a CaM-kinase cascade in Jurkat T human lymphocytes through antigen receptor (CD3) signaling. Total and Ca-independent CaM-kinase IV activities were increased 8-14-fold by anti-CD3 antibody. This CD3-mediated activation involved phosphorylation since the immunoprecipitated CaM-kinase IV from stimulated Jurkat cells could be subsequently inactivated in vitro by protein phosphatase 2A. CaM-kinase IV immunoprecipitated from unstimulated Jurkat cells or CD3-negative mutant Jurkat cells could be activated in vitro 10-40-fold by CaM-kinase kinase purified from rat brain or thymus, whereas CaM-kinase IV from CD3-stimulated wild-type Jurkat cells was only activated to 2-3-fold by exogenous CaM-kinase kinase. CaM-kinase IV activation was triggered by Ca acting through calmodulin since activation could also be elicited by ionomycin treatment, and CD3-mediated activation was blocked by the calmodulin antagonist calmidazolium. These data are consistent with a CaM-kinase cascade in which CaM-kinase IV is activated by a CaM-kinase kinase cascade triggered by elevated intracellular calcium in Jurkat cells.


INTRODUCTION

Ca/calmodulin-dependent protein kinase IV (CaM-kinase IV), (^1)a member of the CaM-kinase family, was first named CaM-kinase-Gr due to its abundance in rat cerebellar granule cells(1) . Subsequently, CaM-kinase IV was found in thymus, spleen, and testis(2) , and it was localized to both the cytoplasm and nucleus(3) . The substrate specificity of CaM-kinase IV has not been well established, but it can phosphorylate in vitro the synthetic peptide syntide-2, synapsin I(1) , Rap-1b(4) , and several transcription factors including serum response factor (5) and cAMP-responsive element binding protein(6, 7) . Recently, it was shown that oncoprotein 18 (Op 18) is a major cytosolic target for CaM-kinase IV in Jurkat cells(8) . Although the function of Op 18 is not clear, its involvement in signal transduction and/or cell cycle control has been suggested(9, 10, 11, 12) .

The primary structure of CaM-kinase IV has been resolved by cDNA cloning from rat (1, 13, 14) and mouse brain (15) and human Jurkat T lymphocytes(16) . CaM-kinase IV is structurally related to CaM-kinase II in its catalytic and regulatory CaM-binding domains(1) . However, the COOH terminus of CaM-kinase IV is highly acidic with polyglutamate stretches, a characteristic of many chromatin-associated proteins(17) , which probably accounts for its monomeric structure. Truncation of CaM-kinase IV at residue 313 produces a CaM-independent kinase(18) , and an autoinhibitory domain within residues 305-321 has been characterized by site-specific mutagenesis(19) .

It was originally thought that CaM-kinase IV is activated by autophosphorylation since the enzyme purified from rat brain and thymus exhibits 10-fold or greater increases in both total (i.e. assayed in the presence of Ca/CaM) and Ca-independent (assayed in the presence of EGTA) activities upon incubation in the presence of Ca/CaM and ATP/Mg(2, 20, 21) . However, recombinant CaM-kinase IV expressed in Escherichia coli(16, 22) or baculovirus/Sf9 cells(18, 19) exhibits very slow and substoichiometric autophosphorylation associated with only a 2-fold activation even after incubation for 1-2 h. It has been demonstrated recently that recombinant CaM-kinase IV can be activated by preincubation with brain extract in the presence of ATP/Mg and Ca/CaM(16, 19, 22) , and a 66-68-kDa CaM-kinase kinase has been purified from rat brain(19, 23) . After activation by CaM-kinase kinase, the ratio of V(max)/K for CaM-kinase IV phosphorylation of the transcriptional activator cAMP-responsive element binding protein is increased 30-fold(7) . Although transfected CaM-kinase IV weakly stimulates transcription of a reporter gene through phosphorylation of cAMP-responsive element binding protein, co-transfection of CaM-kinase IV with the recently cloned CaM-kinase kinase potentiates transcriptional activation by 14-fold(24) . This is good evidence in transfected cells for a CaM-kinase cascade involving CaM-kinase IV, which can regulate transcription.

CaM-kinase IV might be involved in T cell development since it is more abundant in immature thymocytes compared with mature thymocytes and circulating T lymphocytes, and it is not expressed in B lymphocytes or monocytes(25) . In Jurkat T lymphocytes, both Ca-independent and total activities of CaM-kinase IV are maximally increased within 1 min of stimulation of the CD3 receptor(25) . Stimulation of the CD3 receptor is known to activate multiple cellular signaling systems including protein tyrosine kinases, phospholipase C, protein kinase C, and mobilization of IP(3)-sensitive Ca stores (see review in (26) ). Therefore, Jurkat cells seem to provide a good system to study the regulation of CaM-kinase IV in situ. In this paper, we demonstrate that 1) CaM-kinase IV activated through CD3-receptor stimulation or in vitro by CaM-kinase kinase can be inactivated by type 2A protein phosphatase but not by type 1 phosphatase, 2) CaM-kinase kinase purified from rat brain and thymus activates CaM-kinase IV from unstimulated Jurkat cells, and 3) ionomycin activates CaM-kinase IV whereas a calmodulin antagonist blocks activation. We conclude that activation of CaM-kinase IV in response to CD3 receptor stimulation is mediated by Ca/CaM, probably through activation of CaM-kinase kinase.


EXPERIMENTAL PROCEDURES

Cell Culture

Jurkat human leukemic T lymphocyte (clone E6-1) and a CD3-negative mutant (J.RT3-T3.5) were obtained from American Type Culture Collection (Rockville, MD) and maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum and 2 mML-glutamine. Cell viability was monitored by trypan blue exclusion.

Materials

Monoclonal antibody Leu-4 was purchased from Becton Dickinson, and RPMI 1640 and microcystin-LR were from Life Technologies, Inc. Ionomycin and pansorbin were obtained from Calbiochem, and Nonidet P-40, rabbit control IgG, and rabbit anti-mouse IgG were from Sigma. Leupeptin, antipain, and pepstatin A were purchased from Transformation Research, and protein kinase A inhibitor (PKI 5-24) and protein kinase C inhibitor (PKC 19-36) peptides were from Peninsula Laboratories. Calyculin A and calmidazolium were obtained from LC Laboratories, and phenylmethylsulfonyl fluoride and DTT were from Boehringer Mannheim. Syntide-2 (PLARTLSVAGLPGKK) was synthesized and purified as described(27) . The [-P]ATP was from DuPont NEN.

CaM-kinase IV Antibody Production

Rabbit polyclonal anti-human CaM-kinase IV antiserum was raised against the COOH-terminal sequence, (CG)GLAEEKLTVEEA, whose first two residues were introduced to facilitate conjugation to keyhole lympet hemocyanin (Macromolecular Resources, Colorado State University). CaM-kinase IV antibody was purified from rabbit serum by affinity chromatography using the antigen peptide coupled to Sulfolink gel (Pierce) according to the manufacturer's instructions.

Proteins

The alpha-isoform of recombinant protein phosphatase type 1 catalytic subunit (PP1C) was a kind gift from Dr. Anna DePaoli-Roach (Indiana University, IN), and human red blood cell protein phosphatase 2A (PP2A) was purchased from Upstate Biotechnology Inc. Calmodulin (CaM) was purified from bovine brain(28) . Recombinant mouse CaM-kinase IV was purified from Sf9 cells by 50% ammonium sulfate fractionation of the 100,000 times g supernatant followed by chromatography on CaM-Sepharose as described previously (6) . Purified rat brain CaM-kinase kinase was provided by Dr. H. Tokumitsu and was purified as described(19) . Rat thymus CaM-kinase kinase was also purified using the same protocols including batch adsorption with P-11 (Whatman), ammonium sulfate fractionation by 50% saturation, chromatography on Q-Sepharose (Pharmacia Biotech Inc.), CaM-Sepharose (Pharmacia), Affi-Gel blue (Bio-Rad), and heparin-agarose (Sigma). Protein concentrations were determined by the Bradford method using -globulin (Bio-Rad) as a standard.

Western Blotting and Immunoprecipitation

Jurkat cells (3 times 10^7 cells) were collected by brief centrifugation in a microfuge and resuspended in 350 µl of ice cold homogenization buffer containing 50 mM HEPES, pH 7.5, 2 mM EDTA, 2 mM EGTA, 5 mM DTT, 0.5% Nonidet P-40, 10 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 0.5 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, 10 µg/ml each of N-p-tosyl-L-lysine chloromethyl ketone, leupeptin, pepstatin A, soybean trypsin inhibitor, antipain, and aprotinin. After incubation on ice for 15 min, the homogenates were centrifuged at 12,000 times g for 30 min at 4 °C. Eight µl of supernatant was used for Western blotting, and 300 µl of supernatant was used for immunoprecipitation of CaM-kinase IV. For immunoblotting, proteins were separated on a 10% SDS-polyacrylamide gel and transferred electrophoretically to nitrocellulose membrane (Schleicher & Schuell). The membrane was blocked for 1 h at room temperature with TBST (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) containing 5% nonfat powder milk, and it was then incubated with anti-CaM-kinase IV antibody at 2 µg/ml in the blocking buffer for 1 h. After washing three times with TBST for 10 min, the membrane was incubated with donkey anti-rabbit IgG conjugated with horseradish peroxidase (Amersham Corp.) at 1:2000 dilution for 1 h in the blocking buffer. The membrane was washed extensively, and the bound antibody was visualized by chemiluminescence reagent (DuPont NEN).

For immunoprecipitation of CaM-kinase IV, supernatant was precleared for 1 h with pansorbin coated with rabbit control IgG (Sigma), and the supernatant was then incubated with 9.6 µg of CaM-kinase IV antibody for 1 h at 4 °C. Antigen-antibody complex was precipitated with pansorbin for 30 min on ice. The immunoprecipitates were washed three times with TBS plus 0.3 M NaCl followed by one wash with 50 mM HEPES and 0.1 mM EDTA. Final immunoprecipitates were resuspended in 40 µl of kinase dilution buffer consisting of 50 mM HEPES, pH 7.5, 1 mg/ml bovine serum albumin and 10% ethylene glycol, and 5 µl were used for syntide-2 kinase assay as described below.

Protein Phosphatase Treatment

CaM-kinase IV immunoprecipitated from Leu-4-treated Jurkat cells or recombinant CaM-kinase IV activated in vitro with CaM-kinase kinase was incubated with PP1C (100 milliunits/ml) or PP2A (3.85 milliunits/ml) in a 25-µl reaction mixture containing kinase dilution buffer plus 0.2 mM MnCl(2) and 0.2% beta-mercaptoethanol in the presence or absence of 0.1 µM calyculin. After 20 min at 30 °C, the reactions were stopped with calyculin to a final concentration of 0.1 µM. Five microliters were used to measure syntide-2 kinase activity. One unit of phosphatase is the amount of enzyme that releases 1 nmol of P0(4) from P-phosphorylase a (1 mg/ml)/min at 30 °C.

Enzyme Assays

For measuring CaM-kinase kinase activity in Jurkat cell extracts, cell lysates (0.5 mg/ml) were incubated in a 25-µl reaction mixture containing 50 mM HEPES, 2 mM DTT, 5 µM baculovirus-expressed CaM-kinase IV, 2 mM CaCl(2), 10 µM CaM, 10 mM magnesium acetate, 0.4 mM ATP, and 1 µM microcystin-LR for 5 min at 30 °C. Preliminary experiments established that these conditions were within the linear time course of CaM-kinase IV activation. Reactions were stopped by diluting 25-fold with the stopping buffer (50 mM HEPES, pH 7.5, 1 mM EDTA, and 10% ethylene glycol), and then 5 µl were used to determine CaM-kinase IV activity.

For CaM-kinase kinase treatment of Jurkat cell CaM-kinase IV, 5 µl of immunoprecipitated CaM-kinase IV was incubated with purified rat brain or thymus CaM-kinase kinase for 10 min at 30 °C in a 25-µl reaction mixture containing 50 mM HEPES, 2 mM DTT, 0.2 mg/ml bovine serum albumin, 2% ethylene glycol, 1 mM CaCl(2), 5 µM CaM, 10 mM magnesium acetate, 0.4 mM ATP, and 1 µM microcystin-LR. The reaction was terminated with 500 µl of the stop buffer followed by immediate centrifugation in a microfuge for 5 min at 4 °C. The CaM-kinase IV immunoprecipitates were resuspended in 25 µl of kinase dilution buffer. Five microliters were used for kinase assay. All assays were performed in duplicate.

CaM-kinase IV activity was measured by P-incorporation into syntide-2 in a 25-µl reaction mixture containing 50 mM HEPES, pH 7.5, 2 mM DTT, 40 µM syntide-2, 0.2 mM [-P]ATP (500-1,000 cpm/pmol), 10 mM magnesium acetate, 5 µM PKI 5-24, 2 µM PKC 19-36 inhibitor peptides, 1 µM microcystin-LR, and either 1 mM EGTA (for Ca/CaM-independent activity) or 0.8 mM CaCl(2) and 1 µM CaM (for total activity). The reaction proceeded for 10 min (unless otherwise indicated) at 30 °C and was then terminated by spotting a 15-µl aliquot onto phosphocellulose P81 paper (Whatman) followed by washing with 75 mM phosphoric acid(29) .

Phosphorylation of Recombinant CaM-kinase IV by Rat Thymus CaM-kinase Kinase

Baculovirus-expressed CaM-kinase IV (0.38 µg) was phosphorylated by rat thymus CaM-kinase kinase (0.14 µg) for 60 min at 30 °C in a 15-µl reaction mixture containing 50 mM HEPES, pH 7.5, 2 mM DTT, 4 mM magnesium acetate, 0.1 mM [-P]ATP (specific activity of 10,000 cpm/pmol), in the presence of 1 mM EGTA or 4 mM CaCl(2) and 2 µM CaM. Reactions were stopped with SDS-sample buffer, and proteins were separated on a 10% SDS-polyacrylamide gel. Phosphorylated bands were visualized by autoradiography.


RESULTS

Activation of CaM-kinase IV in Jurkat T Lymphocytes

Jurkat E6-1 cells were treated with an anti-CD3 monoclonal antibody (Leu-4) for 1 min, and kinase activities in the cell extract were assayed for Ca-independent activity (measured in the presence of EGTA) and total kinase activity (measured in the presence of Ca/CaM) using syntide-2 as a substrate. In comparison with nonstimulated cells, CD3-treatment resulted in less than 2-fold increases in both Ca-independent and total syntide-2 kinase activities (data not shown). This result does not indicate whether only CaM-kinase IV has been activated since multiple protein kinases can phosphorylate syntide-2. For example, activation of CaM-kinases I and II would increase total and Ca-independent activities, respectively, whereas activation of CaM-kinase IV enhances both total and Ca-independent activities. A specific substrate for CaM-kinase IV is not available, so it was necessary to assay CaM-kinase IV in an immunoprecipitate as was done in the original Jurkat cell study(25) . Toward this end we generated a CaM-kinase IV antibody using a synthetic peptide antigen from a unique COOH-terminal sequence of human CaM-kinase IV (residues 436-447). This sequence is not conserved in CaM-kinases I (30, 31) or CaM-kinase II (32, 33, 34, 35) , and a Western blot of Jurkat cell extracts detects specific interaction with a doublet at 59 and 60 kDa, the known isoforms of CaM-kinase IV (Fig. 1A, lane 1). The immunoreactive band at 55 kDa probably represents a nonspecific interaction with the secondary antibody, as this polypeptide was not immunoprecipitated by anti-CaM-kinase IV antibody (Fig. 1A, lane 2). Therefore, the syntide-2 assay in the immunoprecipitate is specific for CaM-kinase IV.


Figure 1: Activation of Jurkat cell CaM-kinase IV via CD3 receptor stimulation. A, Jurkat E6-1 cell lysate supernatants were subjected to Western blot analyses using antibody to CaM-kinase IV before (lane 1) and after (lane 2) immunoprecipitation with anti-CaM kinase IV. B, Jurkat cells (3 times 10^7) were treated for the indicated times with 0.3 µg/ml anti-CD3 (Leu-4) in the presence of 25 µg/ml rabbit anti-mouse IgG. Cells were centrifuged and lysed, and CaM-kinase IV was immunoprecipitated with anti-CaM kinase IV. CaM-kinase IV activity was assayed in the presence (black bars) and absence (gray bars) of Ca/CaM (see ``Experimental Procedures''). C, Jurkat E6-1 and J.RT3-T3.5 cells were treated with 25 µg/ml rabbit anti-mouse IgG in the presence or absence of 0.3 µg/ml Leu-4 for 1 min, and CaM-kinase IV activity was measured in the presence of EGTA (gray bars) or Ca/CaM (black bars). Error bars indicate standard deviation from five independent experiments in duplicate.



When immunoprecipitates from unstimulated Jurkat cells were assayed for CaM-kinase IV activity, there were very low total and Ca-independent activities (Fig. 1, B and C). Stimulation with anti-CD3 resulted in elevated total and Ca-independent CaM-kinase IV activities, which were maximal within 1 min of stimulation (Fig. 1B) and at 0.2 µg/ml of anti-CD3 (data not shown). In CD3-stimulated cells the Ca-independent activity was enhanced approximately 20-fold, and the total activity was enhanced about 8-fold (Fig. 1C, left panel). The ratio of Ca-independent to total activity was increased from <0.2 in unstimulated cells to 0.3-0.4 in stimulated cells. To demonstrate that CaM-kinase IV was activated through the T cell receptor (TCR)-CD3 complex, a mutant Jurkat clone J.RT3-T3.5 (CD3-negative) was also treated with anti-CD3 (Fig. 1C, right panel). This mutant Jurkat clone lacks the beta-subunit of TCR; therefore, no TCR-CD3 complex is assembled on the cell surface(36) . CaM-kinase IV activity was not increased after anti-CD3 treatment of this mutant clone, and this was not due to a lower level of CaM-kinase IV protein in these cells, since Western immunoblotting revealed a similar level of CaM-kinase IV in this Jurkat cell line (data not shown). These results demonstrate that CD3-induced activation of CaM-kinase IV in Jurkat required signaling through TCR-CD3.

Activation of Immunoprecipitated Jurkat CaM-kinase IV by Purified CaM-kinase Kinase

CaM-kinase kinase has been purified from rat brain(19, 23) , and it can activate recombinant CaM-kinase IV in vitro, resulting in a 20-fold increase in Ca-independent activity and a 10-fold increase in total activity. If the CD3-mediated activation of CaM-kinase IV were due to phosphorylation by CaM-kinase kinase, then CaM-kinase IV from unstimulated cells should be activated in vitro by exogenous CaM-kinase kinase, whereas activated CaM-kinase IV from stimulated cells should be much less responsive to subsequent in vitro activation by CaM-kinase kinase.

To test this hypothesis, CaM-kinase IV was immunoprecipitated from both control and CD3-stimulated Jurkat cells and then incubated with or without purified rat brain CaM-kinase kinase (19) prior to the assay for CaM-kinase IV activities. In vitro incubation with CaM-kinase kinase enhanced both Ca-independent and total activities of CaM-kinase IV from unstimulated cells by 40- and 8-fold, respectively (Fig. 2A, Control). This in vitro increase in total CaM-kinase IV activity was about 2-fold greater than the activation due to CD3-treatment in this particular experiment (Fig. 2A; Stimulated, CaM KK -). When CaM-kinase IV from CD3-stimulated Jurkat cells was treated in vitro with rat brain CaM-kinase kinase, the final activity was approximately the same as achieved by the in vitro activation of CaM-kinase IV from control cells. The fact that CD3-stimulation and subsequent in vitro treatment by CaM-kinase kinase gave nonadditive activation of CaM-kinase IV suggests that CD3-activation in the Jurkat cells may use the same mechanism(s) as activation by CaM-kinase kinase in vitro. Note that there was little if any activation by in vitro incubation in the absence of added CaM-kinase kinase, consistent with a lack of Jurkat CaM-kinase IV activation by autophosphorylation.


Figure 2: Activation of Jurkat CaM-kinase IV by rat brain CaM-kinase kinase. Five microliters of CaM-kinase IV immunoprecipitated from wild-type (panel A) or CD3-negative (panel B) Jurkat cells, which had been treated with 25 µg/ml of rabbit anti-mouse IgG alone (Control) or with 0.3 µg/ml Leu-4 (Stimulated) as described in the Fig. 1legend were preincubated for 10 min at 30 °C with Ca/CaM and ATP/Mg in the absence(-) or presence (+) of purified rat brain CaM-kinase kinase (0.26 µg/ml). CaM-kinase IV activity was measures as described under ``Experimental Procedures.'' Dotted bars indicate Ca/CaM-independent activity, and filled bars indicate total activity.



CaM-kinase IV from CD3-negative Jurkat cells was also fully activated in vitro by CaM-kinase kinase (Fig. 2B), indicating that the J.RT3-T3.5 cells contained normal CaM-kinase IV and that the inability of CD3-stimulation to activate CaM-kinase IV in these cells is due to lack of proper signaling from the cell surface. Therefore, these results suggest that CaM-kinase IV might be directly activated by CaM-kinase kinase in vivo.

Activated CaM-kinase IV Can Be Inactivated by PP2A but Not by PP1

To determine whether the increase in CaM-kinase IV activity in CD3-treated Jurkat cells was in fact due to phosphorylation, immunoprecipitated CaM-kinase IV from CD3-stimulated Jurkat cells was treated with protein phosphatases prior to the kinase assay. It has been previously shown that the in vitro activation of CaM-kinase IV by purified brain CaM-kinase kinase can be reversed by the catalytic subunit of protein phosphatase 2A(19) , and preincubation of the CD3-activated CaM-kinase IV with PP2A reversed the increases in both total and Ca-independent activities of CaM-kinase IV by 80 and 60%, respectively (Fig. 3A). As expected, the effect of PP2A was blocked by the protein phosphatase inhibitor calyculin. Interestingly, protein phosphatase 1 (PP1) was not able to inactivate CaM-kinase IV from CD3-stimulated cells (Fig. 3A); nor was PP1 able to inactivate recombinant CaM-kinase IV activated in vitro by CaM-kinase kinase (Fig. 3B), even though it was active against phosphorylase a (data not shown).


Figure 3: Inactivation of activated CaM-kinase IV by protein phosphatase treatment. A, CaM-kinase IV immunoprecipitated from CD3-stimulated (0.3 µg/ml for 1 min) Jurkat cells was incubated with PP2A (3.85 milliunits/ml) and PP1C (100 milliunits/ml) in the absence or presence of 0.1 µM calyculin A for 20 min at 30 °C. Reactions were stopped with 0.1 µM calyculin and assayed for syntide-2 kinase activity. Dotted bars indicate Ca/CaM-independent activity, and filled bars indicate total activity. Total activity of each non-phosphatase-treated sample was equated to 100% (average value = 2.34 ± 0.8 pmol/min). The results show means and standard deviations from four experiments. B, recombinant CaM-kinase IV (5 µM) was activated with recombinant rat brain CaM-kinase kinase expressed in COS cells(24) , and the samples were incubated with protein phosphatases and assayed for CaM-kinase IV as in panel A. 100% activity = 8.5 pmol/min. For details see ``Experimental Procedures.''



CaM-kinase Kinase Activity in Jurkat Cell Extracts during T Cell Activation

To determine whether CaM-kinase kinase activity is present in Jurkat cells and if it is regulated during T cell activation, Jurkat cells were stimulated with increasing amounts (0-0.8 µg/ml) of anti-CD3 antibody for 1 min. The cell extracts were then tested for CaM-kinase kinase activity in vitro by assaying their ability to activate recombinant CaM-kinase IV. Jurkat cells contained CaM-kinase IV activating activity, but there was no significant change in their CaM-kinase kinase activity in response to CD3 receptor activation (data not shown). In vitro activation of recombinant CaM-kinase IV by the Jurkat cell extract was dependent on Ca/CaM, as no activation occurred in the presence of EGTA (data not shown). Mutant Jurkat clone J.RT3-T3.5 also contained a similar amount of CaM-kinase kinase activity (data not shown).

Purification of CaM-kinase Kinase from Rat Thymus

CaM-kinase kinase was purified from rat thymus by the procedure used for purification of the rat brain kinase(19) . Like the rat brain enzyme, thymus CaM-kinase kinase did not bind phosphocellulose P-11, and it was eluted at positions similar to rat brain kinase during various column chromatography steps including Q-Sepharose, CaM-Sepharose, Affi-Gel blue, and heparin-agarose (data not shown). The molecular mass of the thymus CaM-kinase kinase was about 68 kDa as determined by Western analysis (data not shown) using an antibody against a highly conserved protein kinase motif(24) . The fact that the enzyme can bind CaM-Sepharose in a Ca-dependent manner indicates that rat thymus CaM-kinase kinase is also a Ca/CaM binding protein, and this was further documented by its reactivity toward biotinylated-CaM using the CaM-overlay procedure (data not shown). To determine whether rat thymus CaM-kinase kinase can phosphorylate CaM-kinase IV in vitro, recombinant CaM-kinase IV was incubated with or without thymus CaM-kinase kinase in the presence or absence of Ca/CaM. CaM-kinase IV alone showed weak autophosphorylation in the presence of Ca/CaM (Fig. 4A, lane 6). Thymus CaM-kinase kinase itself did not exhibit detectable autophosphorylation (Fig. 4A, lanes 1 and 2), but it did catalyze strong Ca/CaM-dependent phosphorylation of CaM-kinase IV (lanes 3 and 4).


Figure 4: Phosphorylation and activation of CaM-kinase IV by rat thymus CaM-kinase kinase. A, Sf9-expressed CaM-kinase IV (0.38 µg) was phosphorylated with partially purified rat thymus CaM-kinase kinase (0.14 µg) for 60 min at 30 °C in the presence of 1 mM EGTA (lanes 1, 3, and 5) or 4 mM CaCl(2) and 2 µM CaM (lanes 2, 4, and 6). The reactions were stopped by adding 5 times SDS-sample buffer, and the reactions were then separated by 10% SDS-polyacrylamide gel electrophoresis. Top panel, Coomassie stain. Bottom panel, P-incorporation was visualized by autoradiography. Lanes 1 and 2, CaM-kinase kinase (CaM KK) alone; lanes 3 and 4, CaM-kinase IV and CaM-kinase kinase; lanes 5 and 6, CaM-kinase IV alone. B, CaM-kinase IV immunoprecipitated from unstimulated Jurkat E6-1 cells was incubated without(-) or with (+) 0.96 µg/ml CaM-kinase kinase partially purified from rat thymus in the presence of Mg/ATP and Ca/CaM as in Fig. 2, and CaM-kinase IV activity was then measured. Open bars indicate Ca/CaM-independent activity, and filled bars indicate total activity. The data represent means and standard errors from four experiments.



Next, we tested whether thymus CaM-kinase kinase could activate CaM-kinase IV in the immunoprecipitates from unstimulated and CD3-stimulated Jurkat cells. As shown in Fig. 4B, preincubation of CaM-kinase IV immunoprecipitated from unstimulated Jurkat cells with thymus CaM-kinase kinase led to strong activation of CaM-kinase IV.

Activation of CaM-kinase IV by Ionomycin and Inhibition of CD3-mediated Activation by Calmidazolium

It is known that stimulation of the CD3 receptor results in elevation of intracellular Ca from IP(3)-sensitive stores (37) as well as activation of other signaling pathways. To determine whether an increase in intracellular concentration of Ca is sufficient for CaM-kinase IV activation, Jurkat cells were treated with increasing amounts of ionomycin, and CaM-kinase IV was immunoprecipitated and assayed for kinase activity. Ionomycin increased CaM-kinase IV activity in a dose-dependent manner, reaching a plateau value at 5 µM (Fig. 5). Immunoprecipitated CaM-kinase IV from ionomycin-treated cells could be activated an additional 2-3-fold by in vitro treatment with CaM-kinase kinase (data not shown), similar to the CD3-activated CaM-kinase IV in Fig. 2. Ionomycin also activated CaM-kinase IV in the CD3-deficient Jurkat cells (data not shown), consistent with our results showing the presence of CaM-kinase kinase in this mutant cell line. These results demonstrate that elevated intracellular Ca can activate CaM-kinase IV.


Figure 5: Activation of CaM-kinase IV by ionomycin and inhibition of CD3-mediated activation by calmidazolium. A, Jurkat cells (3 times 10^7 cells/ml) were preincubated for 10 min at 37 °C and then treated with increasing concentrations of ionomycin for 2 min in duplicate. CaM-kinase IV was immunoprecipitated, and Ca/CaM-independent (open circles) and total activities (closed circles) were measured as described under ``Experimental Procedures.'' B, Jurkat cells (2 times 10^7 cells/ml) were preincubated with the indicated amount of calmidazolium for 20 min at 37 °C and then stimulated with 0.3 µg/ml of Leu-4 for 1 min. CaM-kinase IV was immunoprecipitated, and total activity was determined as described above. Two independent experiments in duplicate are shown. 100% activity = 5.2 pmol/min. *, CaM-kinase IV activity from unstimulated cells.



If activation of CaM-kinase IV by phosphorylation in CD3-stimulated Jurkat cells is mediated by CaM-kinase kinase, then this activation should be blocked by the calmodulin antagonist calmidazolium because CaM-kinase kinase is a Ca/CaM-dependent enzyme(19, 24) . Preincubation of the Jurkat cells with calmidazolium resulted in complete inhibition of the CD3-mediated activation (Fig. 5B). Since CaM-kinase IV also binds Ca/CaM, one cannot conclude that the sole effect of calmidazolium was on CaM-kinase kinase. It is possible that phosphorylation of CaM-kinase IV requires binding of Ca/CaM to both CaM-kinase kinase and CaM-kinase IV. However, this seems unlikely since a Ca/CaM-independent mutant of CaM-kinase IV still requires Ca/CaM for activation by CaM-kinase kinase(19) , and the activating phosphorylation site in CaM-kinase IV is located in the catalytic domain(38) , far removed in the primary sequence from the autoinhibitory/CaM-binding domains.


DISCUSSION

There is accumulating evidence suggesting that some members of the CaM-kinase family, namely CaM-kinase Ia and CaM-kinase IV, are regulated by upstream protein kinases(19, 23, 39, 40) . Unlike CaM-kinase II, whose total activity can be fully stimulated by Ca/CaM (see review in (41) ), recombinant CaM-kinase IV has a very low specific activity in the presence of Ca/CaM and requires phosphorylation by CaM-kinase kinase for full activity. CaM-kinase kinases for CaM-kinase Ia (39, 40) and CaM-kinase IV (19, 23) have been purified from pig brain and rat brain, respectively, and characterized. Both require ATP/Mg and Ca/CaM for their activities, but CaM-kinase Ia kinase is a 52-kDa whereas CaM-kinase IV kinase is a 66-68-kDa protein. Recently, a 68-kDa rat brain CaM-kinase kinase has been cloned, and the expressed CaM-kinase kinase can catalyze Ca/CaM-dependent activation of both CaM-kinases Ia and IV but not CaM-kinase II(24) . The recombinant CaM-kinase kinase increased both Ca-independent and total activities of CaM-kinase IV, whereas it enhanced only total activity of CaM-kinase Ia. Similar results have been obtained with the purified pig brain CaM-kinase I kinase(38) . The present study was initiated to determine if a CaM-kinase cascade involving CaM-kinase IV and CaM-kinase kinase can be demonstrated in cultured cells. Jurkat cells were chosen because they had previously demonstrated activation of CaM-kinase IV by CD3 receptor stimulation(25) . We were able to reproduce their results, and our study provides support for the hypothesis that CD3 receptor stimulation activates CaM-kinase IV through phosphorylation by CaM-kinase kinase.

Because there is no specific assay for CaM-kinase IV, it was important to first establish that our assay detected only CaM-kinase IV. The most likely interference in the assay would come from CaM-kinase II since it exhibits high specific activity for syntide-2 phosphorylation, but such interference is unlikely since the antibody we used to immunoprecipitate CaM-kinase IV does not show cross-reactivity with either CaM-kinase I or II in the Jurkat cell extract (Fig. 1A). Furthermore, CaM-kinase II is not known to increase its total activity upon phosphorylation, and its increase in Ca-independent activity upon autophosphorylation is reversed by both protein phosphatases 1 and 2A (42, 43, 44) .

Our results indicate that activation of CaM-kinase IV upon CD3-stimulation of Jurkat cells is mediated by CaM-kinase kinase. CaM-kinase kinase activity is present in Jurkat cells, and it was purified from thymus (Fig. 4) and shown to have properties similar to the purified rat brain enzyme. Second, the activation of CaM-kinase IV by CD3-stimulation of Jurkat cells and by in vitro activation with CaM-kinase kinase were nonadditive (Fig. 2), indicating that the same or overlapping activation mechanisms are involved. Third, CaM-kinase IV activated in Jurkat cells by CD3-stimulation (Fig. 3A) or recombinant CaM-kinase IV activated in vitro with CaM-kinase kinase (Fig. 3B) was subsequently inactivated in vitro by PP2A but not by PP1. This unusual specificity for PP2A for dephosphorylation is consistent with the same site being phosphorylated by CaM-kinase kinase in vitro and by CD3-stimulation in the Jurkat cells. Last, activation for CaM-kinase IV in Jurkat cells can be stimulated by Ca mobilization (i.e. ionomycin, Fig. 5A) and blocked by a calmodulin antagonist (Fig. 5B). These observations support the hypothesis that CD3 receptor stimulation activates CaM-kinase IV through an upstream cascade involving CaM-kinase kinase.

The mechanism by which CD3 stimulation activates CaM-kinase kinase is probably due to elevation of intracellular Ca(37) . Consistent with this hypothesis, CD3 stimulation does not appear to generate a stable activation of CaM-kinase kinase, such as phosphorylation, that survives cell lysis (data not shown). Furthermore, CD3 stimulation does not fully activate CaM-kinase IV, as subsequent in vitro phosphorylation by CaM-kinase kinase results in an additional 2-3-fold increase in total CaM-kinase IV activity (Fig. 2A). The most likely explanation is that opposing protein phosphatases limit the extent and duration of activation since activation is maximal around 1 min and returns to basal values at 5 min (Fig. 1B). This temporal pattern would be consistent with high activity of endogenous protein phosphatases, but we have been unsuccessful in attaining further activation or prolonging the duration of activation by treatment with 0.1-0.5 µM okadaic acid, a concentration that should strongly inhibit protein phosphatase 2A. Whereas protein phosphatase 2A reverses the activation of CaM-kinase IV in vitro (Fig. 3), it is possible that the okadaic acid-insensitive phosphatase 2C may reverse the activation in Jurkat cells(45) .

In summary, all of our data is consistent with the hypothesis that CD3 receptor stimulation activates CaM-kinase IV through phosphorylation by CaM-kinase kinase. Further studies will be required to determine whether this is simply the consequence of elevated intracellular Ca or if more complicated signaling pathways are also involved. The latter possibility would be appealing, since it is puzzling why two consecutive steps in a kinase cascade should both be controlled by Ca/CaM. One possibility is that activation of CaM-kinase IV by CaM-kinase kinase requires convergence of signaling pathways in addition to elevated intracellular Ca. Another possibility is that activation of CaM-kinase kinase requires either higher concentrations of Ca or a different pool of Ca than does simple activation CaM-kinase IV by Ca/CaM alone. This would allow selective and transient partial activation of CaM-kinase IV by Ca/CaM alone under some circumstances and stronger activation through phosphorylation by CaM-kinase kinase in response to other agonists. Furthermore, activation of CaM-kinase IV by CaM-kinase kinase has the potential for prolonged activation if the appropriate protein phosphatase activity is low, since the phosphorylated CaM-kinase IV has significant Ca-independent activity. Of course, it is possible that CaM-kinase kinase might regulate currently unidentified substrates that themselves are not Ca/CaM-dependent. Their phosphorylation by CaM-kinase kinase would make them Ca-responsive. These speculative possibilities will require further experimentation.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. This work was supported by National Institutes of Health Grant DK44239.

§
To whom correspondence should be addressed: Vollum Institute L-474, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Rd., Portland, OR 97201-3098. Tel.: 503-494-6931; Fax: 503-494-6934; soderlit@OHSU.EDU.

(^1)
The abbreviations used are: CaM-kinase, Ca/CaM-dependent protein kinase; CaM, calmodulin; DTT, dithiothreitol; TCR, T cell receptor.


ACKNOWLEDGEMENTS

We thank Drs. Hervé Enslen and Hiroshi Tokumitsu from our laboratory for helpful discussions and advice and Dr. Talal Chatila (Washington University) for assistance in setting up the Jurkat cell system.


REFERENCES

  1. Ohmstede, C.-A., Jensen, K. F., and Sahyoun, N. E. (1989) J. Biol. Chem. 264, 5866-5875 [Medline]
  2. Frangakis, M. V., Chatila, T., Wood, E. R., and Sahyoun, N. (1994) J. Biol. Chem. 266, 17592-17596 [Medline]
  3. Jensen, K. F., Ohmstede, C.-A., Fisher, R. S., and Sahyoun, N. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2850-2853 [Medline]
  4. Sahyoun, N., McDonald, O.-B., Farrel, F., and Lapetina, E. G. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2643-2647 [Medline]
  5. Misra, R. P., Bonni, A., Miranti, C. K., Rivera, V. M., Sheng, M., and Greenberg, M. E. (1994) J. Biol. Chem. 269, 25483-25493 [Medline]
  6. Enslen, E., Sun, P., Brickey, D., Soderling, S. H., Klamo, E., and Soderling, T. R. (1994) J. Biol. Chem. 269, 15520-15527 [Medline]
  7. Enslen, E., Tokumitsu, H., and Soderling, T. R. (1995) Biochem. Biophys. Res. Comm. 207, 1038-1043
  8. Marklund, U., Larsson, N., Brattsand, G., Osterman, O., Chatila, T. A., and Gullberg, M. (1994) Eur. J. Biochem. 225, 53-60 [Medline]
  9. Hanash, S. M., Strahler, J. R., Kuick, R., Chu, E. H. Y., and Nichols, D. (1988) J. Biol. Chem. 263, 12813-12815 [Medline]
  10. Hailat, N., Strahler, J. R., Melham, R. F., Zhu, X.-X., Brodeur, G., Seeger, R. C., Reynold, C. P., and Habash, S. M. (1990) Oncogene 5, 1615-1618 [Medline]
  11. Brattsand, G., Roos, G., Marklund, U., Ueda, H., Landberg, G., Nanberg, E., Sideras, P., and Gullberg, M. (1993) Leukemia 7, 569-579 [Medline]
  12. Roos, G., Brattsand, G., Landberg, G., Larsson, U., Marklund, U., and Gullberg, M. (1993) Leukemia 7, 1538-1546 [Medline]
  13. Means, R. A., Cruzalegui, F., LeMaguresse, B., Needleman, D., Slaughter, G., and Ono, T. (1991) Mol. Cell. Biol. 11, 3960-3971 [Medline]
  14. Sakagami, H., and Kondo, H. (1993) Mol. Brain Res. 19, 215-218
  15. Jones, D. A., Glod, J., Wilson-Shaw, D., Hahn, W. E., and Sikela, J. M. (1991) FEBS Lett. 289, 105-109 [Medline]
  16. Kitani, T., Okuno, S., and Fujisawa, H. (1994) J. Biochem. (Tokyo) 115, 637-640
  17. Earnshaw, W. C. (1987) J. Cell Biol. 105, 1479-1482 [Medline]
  18. Cruzalegui, F. H., and Means, A. R. (1993) J. Biol. Chem. 268, 26171-26178 [Medline]
  19. Tokumitsu, H., Brickey, D. A., Glod, J., Hidaka, H., Sikela, J., and Soderling, T. R. (1994) J. Biol. Chem. 269, 28640-28647 [Medline]
  20. Kameshita, I., and Fujisawa, H. (1993) J. Biochem. (Tokyo) 113, 583-590
  21. McDonald, O.-B., Merrill, B. M., Bland, M. M., Taylor, L. C. E., and Sahyoun, N. (1993) J. Biol. Chem. 268, 10054-10059 [Medline]
  22. Okuno, S., and Fujisawa, H. (1993) J. Biochem. (Tokyo) 114, 167-170
  23. Okuno, S., Kitani, T., and Fujisawa, H. (1994) J. Biochem. (Tokyo) 116, 923-930
  24. Tokumitsu, H., Enslen, E., and Soderling, T. R. (1995) J. Biol. Chem. 270, 19320-19324 [JBC][Medline]
  25. Hanissian, S. H., Frangakis, M., Bland, M. M., Jawahar, S., and Chatila, T. A. (1993) J. Biol. Chem. 268, 20055-20063 [Medline]
  26. Cardena, M. E., and Heitman, J. (1995) Adv. Second Messenger Phosphoprotein Res. 30, 281-298
  27. Brickey, D. A., Colbran, R. J., Fong, Y. L., and Soderling, T. R. (1990) Biochem. Biophys. Res. Commun. 173, 578-584 [Medline]
  28. Gopalakrishna, R., and Anderson, W. B. (1982) Biochem. Biophys. Res. Commun. 104, 830-836 [Medline]
  29. Roskoski, R., Jr. (1983) Methods Enzymol. 99, 3-6 [Medline]
  30. Picciotto, M. R., Czernik, A. J., and Nairn, A. C. (1993) J. Biol. Chem. 268, 26512-26521 [Medline]
  31. Cho, F. S., Phillips, K. S., Bogucki, B., and Weaver, T. E. (1994) Biochim. Biophys. Acta 1224, 156-160
  32. Bennett, M. K., and Kennedy, M. B. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1794-1798 [Medline]
  33. Hanley, R. M., Means, A. R., Ono, T., Kemp, B. E., Burgin, K. E., Waxham, N., and Kelly, P. T. (1987) Science 237, 293-297 [Medline]
  34. Bulleit, R. F., Bennette, M. K., Mooloy, S. S., Hurley, J. B., and Kennedy, M. B. (1988) Neuron 1, 63-72 [Medline]
  35. Lin, C. R., Kapiloff, M. S., Durgerian, S., Tatemoto, K., Russo, A. F., Hanson, P., Schulman, H., and Rosenfeld, M. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5962-5966 [Medline]
  36. Weiss, A., and Stobo, J. D. (1984) J. Exp. Med. 160, 1284-1299 [Medline]
  37. Imboden, J. B., and Stobo, J. D. (1985) J. Exp. Med. 161, 446-456 [Medline]
  38. Selbert, M. A., Anderson, K. A., Huang, Q. H., Goldstein, E. G., Means, A. R., and Edelman, A. M. (1995) J. Biol. Chem. 270, 17616-17621 [JBC][Medline]
  39. Lee, J. C., and Edelman, A. M. (1994) J. Biol. Chem. 269, 2158-2164 [Medline]
  40. Lee, J. C., and Edelman, A. M (1995) Biochem. Biophys. Res. Commun. 210, 631-637 [Medline]
  41. Hanson, P. I., and Schulman, H. (1992) Annu. Rev. Biochem. 61, 559-601 [Medline]
  42. Lai, Y., Nairn, A. C., and Greengard, P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 4253-4257 [Medline]
  43. Schworer, C. M., Colbran, R. J., and Soderling, T. R. (1986) J. Biol. Chem. 261, 8581-8584 [Medline]
  44. Brickey, D. A., Bann, J. G., Fong, Y.-L., Perrino, L., Brennan, R. G., and Soderling, T. R. (1994) J. Biol. Chem. 269, 29047-29054 [Medline]
  45. Frangakis, M. V., Ohmstede, C. A., and Sahyoun, N. (1991) J. Biol. Chem. 266, 11309-11316 [Medline]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.