Defective Survival and Activation of Thymocytes in Transgenic Mice Expressing a Catalytically Inactive Form of Ca2+/Calmodulin-Dependent Protein Kinase IV

Kristin A. Anderson1, Thomas J. Ribar1, Maddalena Illario and Anthony R. Means

Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have generated transgenic mice that express a catalytically inactive form of Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) specifically in thymic T cells. The presence of this protein results in a markedly reduced thymic cellularity, although the distribution of the remaining cells is normal based on evaluation of the CD4 and CD8 cell surface antigens that are used to gauge T cell development. Isolated thymic T cells from the transgenic mice also show a dramatically decreased survival rate when evaluated in culture under conditions that do not favor activation. When challenged with an activating stimulus such as {alpha}-CD3 or a combination of phorbol ester plus ionophore, the cells are severely compromised in their ability to produce the cytokine interleukin-2 (IL-2). Reduction of IL-2 production is secondary to the inability to phosphorylate the cAMP response element binding protein, CREB, and induce expression of the immediate early genes such as Fos B that are required to transactivate the IL-2 promoter. Because transgene expression was regulated by the proximal promoter of the murine lck gene and this promoter is inactivated in T cells that exit the thymus, the mutant hCaMKIV is not present in peripheral T cells. Consequently, T lymphocytes present in the spleen can be activated normally in response to either stimulus mentioned above, demonstrating that the effects of the inactive CaMKIV on activation are reversible. Our results suggest that CaMKIV may represent a physiologically relevant CREB kinase in T cells and that the enzyme is also required to ensure normal expansion of T cells in the thymus. Whereas the pathway responsible for this latter role is yet to be elucidated, it is unlikely to include CREB phosphorylation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Signal transduction cascades mediated by changes in the intracellular concentration of Ca2+ are critical for the activation of T lymphocytes in response to occupancy of the T cell receptor (reviewed in 1 . A rise in intracellular Ca2+ occurs very rapidly and is followed by transcriptional activation of immediate early genes of the fos and jun families. These inducible transcription factors, together with preexisting proteins such as the cytoplasmic component of nuclear factor of activated T cells, are required to transactivate the genes encoding cytokines such as interleukin-2 (IL-2) (2, 3, 4). One mandatory Ca2+-dependent step that has been identified is the activation of the Ca2+/calmodulin-dependent protein phosphatase 2B, also known as calcineurin (5). Activation of the phosphatase results in dephosphorylation of the cytoplasmic component of nuclear factor of activated T cells, which allows it to enter the nucleus and heterodimerize with members of the inducible transcription factors (6, 7). As this is the step that is blocked by the immunosuppressive drugs cyclosporin A and FK506 (8), it has received much attention. However, it is clear that other Ca2+-mediated events that impact T cell activation must also exist.

One such event is creation of an anergic state that occurs when T cells are treated with a single reagent that results in Ca2+ influx rather than with a costimulatory signal involving activation of the T cell receptor (9). One of the defects in an anergic cell is blocked transcription of the IL-2 gene. This state can be mimicked by transfection of a constitutively active form of Ca2+/calmodulin-dependent protein kinase II (CaMKII) into the Jurkat T cell line (10, 11). These data suggest that CaMKII is unlikely to represent a target of Ca2+ that is required for T cell activation. That such a target must exist is demonstrated by the fact that, in Jurkat cells, activation of reporter genes containing AP-1-binding sites such as those required for activation of fos and jun family member genes occurs in the presence of immunosuppressive drugs and yet still requires Ca2+ (12). Activation of AP-1 genes requires members of the cAMP response element binding protein (CREB) family (13), and these proteins must be phosphorylated on a specific serine residue (Ser-133 in the case of CREB) (14) to become competent as transcriptional activators. The importance of CREB to T cell activation was revealed by Barton et al. (15), who used the CD2 promoter to target expression of a mutant form of CREB in which Ser-133 was changed to Ala specifically to T cells in transgenic mice. Cells from these animals could not be activated by costimulation through the T cell receptor and produced neither immediate early gene products nor IL-2. The interpretation of these results was that the mutant form of CREB would dimerize with CREB or other binding partners. However, since the mutant form could not be phosphorylated, dimers containing this protein could not function as transactivators. Because phosphorylation is not required for dimerization or DNA binding, the mutant protein appeared to act on transcriptional activation in a dominant/negative manner.

Because T cell activation requires CREB and CREB must be phosphorylated on Ser-133 to function as a transcriptional activator, identification of the physiologically relevant protein kinase would be of considerable interest. Because transcription mediated by AP-1 sequences requires CREB and Ca2+, we suspected that one enzyme responsible for CREB phosphorylation in T cells might be a Ca2+-dependent protein kinase. A logical candidate for this enzyme is the Ca2+/calmodulin-dependent protein kinase IV (CaMKIV). This enzyme is present in T cells (16, 17), is localized in the nucleus (18), and has been shown to phosphorylate CREB specifically on Ser-133 both in vitro and when overexpressed in cultured cell lines (19, 20, 21). In addition, CaMKIV will activate reporter gene constructs that are driven by AP-1 sequences in Jurkat cells (22, 23). Support for this contention comes from the work of Bito et al. (24), who suggested that CaMKIV mediates CREB phosphorylation that is required for the delayed phase of long-term potentiation in cultured hippocampal neurons.

To test the possibility that CaMKIV catalyzes CREB phosphorylation in T lymphocytes, we generated transgenic mice that express a catalytically inactive form of CaMKIV only in T cells when resident in the thymus. Thymic lymphocytes from these animals exhibit striking developmental and T cell receptor-mediated signaling defects. The thymic T cells show dramatic impairment in the ability to phosphorylate CREB, induce transcription of the immediate early gene Fos B, and produce IL-2. Since the transgene is driven by the murine proximal lck promoter and this promoter has been silenced in T cells that exit the thymus (25, 26), splenic T cells do not contain the mutant hCaMKIV protein and regain the ability to signal normally. These data provide compelling evidence that CaMKIV may be an important Ca2+ target required for activation of T cells.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of Transgenic Mice
Figure 1AGo presents diagrammatic representations of the constructs used to produce transgenic mice. The targeting construct is regulated by the proximal promoter of the murine lck gene and includes sequences of the human GH gene at the 3'-end to ensure recognition by the splicing machinery. This construct has been shown to direct high level expression of cDNAs cloned between the lck and human (h) GH sequences exclusively in thymic T cells (25, 27). The diagram on the left illustrates the CaMKIV construct. A single mutation was introduced at Lys-75 (Lys to Glu) of the human CaMKIV cDNA that renders the protein catalytically inactive (23, 28). On the right side of Fig. 1AGo is shown the construct made to produce rat calspermin. Calspermin is a testis-specific calmodulin-binding protein that is composed of the C-terminal 169 amino acids of CaMKIV (29) and is generated from the CaMKIV gene by utilization of a promoter found in the penultimate intron of the gene (30, 31). Since the kinase-inactive CaMKIV retains its high affinity for Ca2+/calmodulin, the calspermin-containing mice were intended to distinguish between any possible dominant/negative effects of the mutant hCaMKIV and effects that might be produced as a consequence of nonspecific Ca2+/calmodulin sequestration. We have demonstrated that both CaMKIV and calspermin are localized predominantly in the nucleus (data not shown).



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Figure 1. Generation of Transgenic Mice

A, Diagrammatic representation of the two transgenes used to generate transgenic mice. The left panel depicts the hCaMKIV construct and the amino acid substitution used to generate the kinase inactive form. The right panel shows the calspermin construct. B, Southern blot analysis of transgenic mice. The left panel depicts two founding lines of hCaMKIVkin(-) mice, Line I and Line K, while the right panel shows one of three founding lines of the calspermin overexpressing mice. C, Expression of endogenous CaMKIV, hCaMKIVkin(-), and calspermin. The left panel shows the expression of CaMKIV in a control mouse (center lane) and from the two different founding lines (left lane is Line K; right lane is Line I). The center panel shows the expression of calspermin in one of the founding lines of mice [note that the calspermin antibody also recognizes CaMKIV]. The right panel shows expression of the hCaMKIVkin(-) in thymic T cells from Line I transgenic mice utilizing an antibody that only recognizes the human form of CaMKIV. Expression of the hCaMKIV is absent in transgenic splenocytes as well as in control thymic and splenic cells.

 
Genotypic characterization of the transgenic mice is shown by the Southern blots in Fig. 1BGo. The transgenic constructs were generated so that the entire transgene could be removed by digestion with Spe1. This would produce a 6.9-kb fragment from integration of the hCaMKIV transgene and a 5.8-kb fragment from integration of the calspermin transgene. The left panel shows the results of a Southern analysis from DNA extracted from two transgenic (one from Line I and one from Line K) and two control mice. It is readily apparent that only the transgenic mice contain a single fragment of the predicted size that hybridizes to the CaMKIV probe. The right panel shows the equivalent experiment carried out on DNA isolated from one transgenic mouse and three control littermates. Again the single predicted fragment is present only in the DNA from the transgenic mouse.

Western blot analysis was used to confirm that the appropriate protein was produced from the respective transgenes. The left panel of Fig. 1CGo shows the results obtained when an antibody is used that recognizes both the endogenous mouse and transgenically produced human CaMKIV. T cell protein from two transgenic mice flank a similar preparation from a control mouse. It can be seen that the expression level of the transgenic protein is higher in the right lane (Line I) than in the left lane (Line K). Densitometry was used to quantify the difference in protein expression. We find that the amount of immunoreactive CaMKIV in the Line I lane is 4-fold greater than in the control lane. Thus the T cells from Line I produce about a 3-fold molar excess of the mutant hCaMKIV relative to endogenous levels of the authentic enzyme. Animals from Line I were used in all subsequent experiments described in this manuscript. We also prepared an antibody to a synthetic peptide containing the C-terminal 17 amino acids of hCaMKIV that, under our experimental conditions, is specific for the human form of the protein. As can be seen in the far left panel of Fig. 1CGo, only T cells resident in the thymus of the transgenic mouse contained the human protein. The fact that no transgenic product is present in T cells isolated from the spleen of the identical transgenic mouse is consistent with the fact that the proximal lck promoter is not active in T cells that are exported from the thymus (26). Additionally, the absence of the mutant hCaMKIV in splenic T cells implies that this protein must be unstable in these cells.

The center panel of Fig. 1CGo shows that T cells removed from the thymus of one line of transgenic mice produce considerable amounts of calspermin. As there are no amino acids present in calspermin that are not also present in CaMKIV, it has not been possible to produce an antibody that recognizes calspermin but not CaMKIV. In addition, the isoelectric pH of calspermin is 4.0, and consequently this protein does not transfer quantitatively under the conditions usually employed for Western blots (29). However, examination of the blots suggests that the amount of calspermin present is in considerable excess to the amount of endogenous CaMKIV. Unquestionably, the ratio is greater than 3:1. Therefore we feel justified in using these cells to control for any nonspecific effects, such as sequestration of Ca2+/calmodulin, due to the presence of the mutant hCaMKIV.

The Presence of Mutant hCaMKIV in T Cells Results in a Developmental Defect
The most obvious phenotypic consequence resulting from the expression of the mutant hCaMKIV was a large reduction in the size and weight of the thymus (data not shown). This gross phenotype was confirmed by analysis of the number of T cells resident in the thymus. As shown in Fig. 2AGo (left panel) the total number of T cells present in the thymus of transgenic mice is only about 16% of that found in the thymus of nontransgenic littermates or in transgenic mice that express calspermin (n = 14, P < 0.01). The deficit observed in the number of T cells in the thymus is completely normalized when the number of splenic cells is quantified. As shown in the left panel of Fig. 2BGo, no statistically significant difference exits between the number of splenocytes in control, transgenic hCaMKIV mutant [lck-CaMKIV Kin(-)], or transgenic calspermin (lck-calspermin) mice. This normalization in the spleen is entirely consistent with the fact that splenocytes cease expression of the hCaMKIV transgene.



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Figure 2. Thymic Cellularity and Survival of Thymic T Cells Are Impaired in hCaMKIV Mutant Transgenic Mice

A, Thymic cellularity and survival of thymic T cells. Cells were harvested, counted, and plated for survival assays as described in Materials and Methods. The left histogram depicts the cellularity of the thymus from both transgenic lines and a control line of mice (n = 14; P < 0.01). The right histogram shows the rate of survival in culture of these same cells (n = 34; P < 0.01). B, Splenic cellularity and survival of splenocytes that were isolated and treated as described in panel A.

 
One explanation for the decreased cellularity of the thymus could be that maturation of T cells within that organ is defective. To examine this question, we used flow cytometry to quantify the number and distribution of cells, based on the cell surface markers CD4 and CD8. The results of these experiments are shown in Fig. 3Go. Cells were isolated from the thymus or spleen, and an equivalent number of live cells (4 x 106) was aliquoted based on trypan blue exclusion followed by counting in a hemocytometer. Thymic cells are shown in the top row of panels in Fig. 3Go. T cells that do not contain either cell surface marker are called double-negative and are distributed in the lower left quadrant. Double positive cells (CD4/CD8) are found in the upper right quadrant. CD4-positive cells are sorted in the upper left and CD8 positive in the lower right. The numbers in the quadrants represent the percent of the total cells that are scored in that quadrant. It is readily seen that there are no differences in the distribution of T cells in the thymus of hCaMKIV mutant, calspermin, or control mice. The lower row of panels shows the same experiment carried out on splenocytes from the identical mice. Only single positive CD4 or CD8 T cells exit the thymus to become stored in the spleen. Therefore, the quadrants to compare are the upper left (CD4) and lower right (CD8). Again no differences are apparent between any of the three animals. The largest number of cells present in the spleen are B lymphocytes and are scored in the lower left quadrant as they do not express either the CD4 or CD8 surface antigens.



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Figure 3. Distribution of CD4 and CD8 T Cells

Cells were isolated from thymi and spleens of transgenic and control mice and stained for flow cytometry using PE-conjugated anti-CD4 and FITC-conjugated anti-CD8 antibodies as described in Materials and Methods. The upper panel shows the distribution of cells from the thymus whereas the lower panel shows the distribution of cells from the spleen. This figure is representative of the results obtained from six independent experiments.

 
The data in Fig. 3Go suggest that development and maturation of T cells are not impaired in the mice expressing the mutant hCaMKIV. However, the ability to generate or maintain normal numbers of T cells during development has been compromised. This decreased cellularity could result from either a significant defect in proliferation and/or an increased rate of cell death of thymic T cells. Our first attempt to address this question was to employ a cell survival assay. These results for thymic and splenic T cells are presented in the upper and lower right histograms of Fig. 2Go, respectively. We used primary culture conditions such that 50–60% of T cells from the control mice will survive a 30-h incubation. Whereas there is no difference between normal thymic T cells and those expressing calspermin, cells containing the mutant hCaMKIV undergo a much greater rate of spontaneous apoptosis (n = 34,P < 0.01). This defect is not apparent in cells isolated from the spleen, a result consistent with the observation that splenic T cells no longer express the transgene product. Thus, at least one explanation for the decreased cellularity found in the thymus of the mice expressing the mutant form of CaMKIV could be that they die more rapidly, at least in cell culture.

The Presence of Mutant hCaMKIV Results in a T Cell-Signaling Defect
It has been shown that CaMKIV is activated within seconds after engagement of the T cell receptor (17, 23, 32). Similarly, CREB is rapidly phosphorylated in response to a T cell-activating signal (15). Because CaMKIV phosphorylates CREB on Ser-133 (19, 20, 21), we examined whether the T cells that expressed the mutant hCaMKIV might have an altered ability to phosphorylate CREB. The results of a representative experiment are shown in Fig. 4Go. As can be seen in the top left panel, addition of phorbol ester [phorbol-12 myristate-13 acetate (PMA)] and ionomycin (shown as P/I as in the figure) to thymic T cells from normal mice resulted in robust phosphorylation of CREB by 1 min. This signal was maintained for at least 30 min. The Ser-133-phosphorylated form of CREB was selectively examined by the use of an antibody that only recognizes this phosphorylated epitope (33). Similar results were obtained when the cells were stimulated by addition of the anti-CD3 antibody that produces the signal through engagement of the T cell receptor ({alpha}-CD3). The location of the phospho-CREB signal is depicted by an arrow. Note that a faster migrating protein also shows an increased phosphorylation in response to addition of P/I or {alpha}-CD3. This protein has been provisionally identified as activating transcription factor-1 (ATF1), another member of the CREB family of transcription factors because: 1) the phosphorylated form of ATF1 is known to be recognized by the phospho-CREB antibody; 2) it is the same size as ATF1 based on migration in an SDS gel; and 3) ATF1 has also been shown to be present in T cells (15) and can be phosphorylated and activated by CaMKIV (34). The third autoradiogram on the left shows that there was no change in the total amount of CREB during the 30 min of the experiment by use of an antibody that recognizes both phosphorylated and unphosphorylated CREB ({alpha}-CREB).



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Figure 4. CREB Phosphorylation Is Inhibited in T Cells That Express Mutant hCaMKIV

A, Thymocytes and splenocytes from control and lck-CaMKIVkin(-) transgenic mice were either unstimulated (uns) or stimulated with PMA + ionomycin or with {alpha}-CD3 for 1, 5, and 30 min. Cell lysates were then subjected to Western blot analysis using antibody specific for CREB phosphorylated on Ser-133 ({alpha}-pCREB) or antibody recognizing both phosphorylated and unphosphorylated CREB ({alpha}-CREB). The arrow indicates the position of CREB. The faster migrating inducible band is tentatively identified as ATF-1. Shown is one representative experiment. (n = 9). B, Thymocytes and splenocytes from lck-calspermin transgenic mice were either unstimulated or stimulated with PMA + ionomycin for 5 and 30 min. Levels of phosphorylated CREB were then determined as described in panel A. Shown is one representative experiment (n = 2).

 
The top right panels of Fig. 4Go show the results obtained when T cells containing the mutant hCaMKIV were stimulated with P/I or {alpha}-CD3. Very little phosphorylation of CREB was observed at any of the three time points in response to either stimulus. In the case of ATF1, we were unable to demonstrate any increase in phosphorylation. The third autoradiogram shows that equivalent amounts of CREB were present at all time points. Comparison of the {alpha}-CREB blots between the normal and mutant hCaMKIV-containing cells shows that the protein is also present in similar amounts in T cells from both types of mice. The abrogation of CREB phosphorylation characteristic of T cells from mice expressing the mutant hCaMKIV was not observed in T cells containing the calspermin protein. Shown in the middle panels of Fig. 4Go is CREB phosphorylation in splenic T cells in response to 2 {alpha}-CD3. Cells from both control and mutant hCaMKIV mice show similar increases in phosphorylation of CREB. The recovery of a normal response in cells from the transgenic mice is again consistent with the fact that splenocytes no longer contain the mutant hCaMKIV protein. Finally, the bottom panels of Fig. 4Go show that the presence of calspermin does not affect phosphorylation of CREB in response to P/I stimulation. We conclude that the presence of the mutant enzyme must result in a dominant/negative effect on CREB phosphorylation whether the stimulus is physiologically relevant, like {alpha}-CD3, or more general, as elicited by the combination of PMA and ionomycin.

CREB phosphorylation in thymic T cells is required for the activation of the immediate early genes c-fos, Fra-2, and Fos B. (15). Therefore we examined the appearance of Fos-B mRNA in response to T cell stimulation. As shown in Fig. 5Go, stimulation of control T cells with {alpha}-CD3 resulted in transient expression of Fos B mRNA. The time course of this response and the presence of the two mRNA species are identical to the results reported by Barton et al. (15). However, cells that express the mutant hCaMKIV fail to demonstrate an increase in Fos B mRNA in response to stimulation. The Northern blot was stripped and reprobed with a glucose-3-phosphate dehydrogenase (GAPDH) cDNA. The bottom autoradiogram in Fig. 5Go shows that each lane did contain equivalent amounts of RNA. Thus the presence of the mutant hCaMKIV correlates with the inability to phosphorylate CREB and to produce Fos B mRNA.



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Figure 5. Fos B Induction Is Inhibited in T Cells That Express Mutant hCaMKIV

Thymic T cells from control and lck-CaMKIVkin(-) transgenic mice were stimulated with PMA + ionomycin for the indicated time. Total RNA was then extracted and subjected to Northern blot analysis using a Fos B-specific probe. To assess RNA sample loading, the blot was striped and reanalyzed with a probe for GAPDH. Shown is one representative experiment (n = 2).

 
The CREB/AP 1 pathway has been shown to be critical for the production of IL-2 (15). Figure 6AGo shows that whereas a 48-h stimulation of T cells from control or calspermin transgenic mice produce equivalent amounts of IL-2 in response to {alpha}-CD3, very little of the cytokine is made by the cells that contain the mutant hCaMKIV. Because one consequence resulting from T cell activation is an increase in the number of cells that express the IL-2 receptor (15, 35, 36), we also examined the appearance of the {alpha}-chain of the IL-2 receptor (CD25) 30 h after cells were challenged with a mitogenic stimulus. The results, shown in Fig. 6BGo, indicate a similar response of control and calspermin-containing cells but a considerable reduction in cells that express mutant hCaMKIV. Qualitative analysis of the data suggest that the former two cell populations show about a 10-fold increase in the proportion that contain CD25, whereas less than a 3-fold increase is seen in the cells containing mutant hCaMKIV.



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Figure 6. Induction of IL-2 and IL-2R{alpha} Are Inhibited in T Cells That Express Mutant hCaMKIV

A, IL-2 induction. IL-2 secretion from control and lck-CaMKIVkin(-) and lck-calspermin transgenic T cells was quantified after stimulation for 48 h with PMA + ionomycin. At the end of a similar experiment, FACS analysis was used to determine the number of T cells remaining. This number did not differ significantly between cells from control or transgenic mice (50 ± 6%). Values for IL-2 (pg/ml) represent the average ± SD of one representative experiment performed in triplicate (n = 5). B, IL-2R{alpha} (CD25) induction. T cells from control and lck-CaMKIVkin(-) and lck-calspermin transgenic mice were stained for CD25 and analyzed by FACS on a flow cytometer before (unshaded curve) and after stimulation for 30 h with PMA + ionomycin (shaded curve). Shown is one representative experiment (n = 4).

 
It must be pointed out that the data presented in Figs. 4–6GoGoGo are representative experiments. Each experiment was repeated several times with T cells from different transgenic mice. Because of the small numbers of such cells, it was not possible to conduct each experiment on cells from the same animals. Therefore, considerable variation was observed from animal to animal. We believe that this is due to the fact that the T cells contained differing amounts of the mutant hCaMKIV. A consistent observation was that the magnitude of the inhibitory responses was directly proportional to the amount of CaMKIV. In addition, when it was possible to carry out two assays on cells from the same animal, the proportional differences were identical when comparing CREB phosphorylation to IL-2 production or to the decrease in the number of cells expressing CD25. For example, if CREB phosphorylation was decreased by 95%, similar percent decreases were found in either of the other two parameters. Based on these observations, we believe that the four events, CREB phosphorylation, induction of Fos B mRNA, IL-2 production, and the expression of CD25, are related to each other and are coincidentally down-regulated by the presence of the mutant hCaMKIV.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have generated transgenic mice to demonstrate that specific expression of a catalytically inactive form of hCaMKIV in T lymphocytes results in two consequences. The first is a dramatic decrease in the number of T cells in the thymus and in survival of thymic T cells in culture. The second is a severe reduction in the ability of T cells to be activated by signals that are propagated through the T cell receptor or generated in response to the activation of protein kinase C coupled to an increase in the concentration of intracellular Ca2+ (i.e. by PMA and ionomycin). Although we present evidence that is likely to explain the mechanism responsible for the abrogation of signaling, we do not yet understand the mechanism responsible for the decrease in the number of T cells. However, it seems quite likely that the mechanisms responsible for these two phenotypic consequences are different.

The question we set out to answer was whether the target of Ca2+ responsible for the activation of AP1-mediated gene transcription that was independent of the action of the immunosuppressive drugs was CaMKIV. To evaluate this possibility, we chose to use a construct designed by Chaffin et al. (27) that used the proximal promoter of the murine lck tyrosine kinase gene to restrict expression to T lymphocytes resident in the thymus. This promoter was chosen for three reasons. First, it is known to be expressed in all double negative and double positive thymic T cells and has been identified as early as embryonic day 15 (26). This is at least 1 day earlier than the first appearance of CaMKIV (45). Therfore, if the mutant hCaMKIV protein was to act in a dominant/negative manner, the kinetics of appearance of the transgene product relative to the endogenous gene product should be optimal to produce an effect. Second, Alberola-Ila et al. (37) have used this vector to express a catalytically inactive form of the mitogen-activated protein kinase kinase, MEK-1, in T cells that effectively blocked the activation of mitogen-activated protein kinase. This dominant/negative approach resulted in the selective inhibition of positive selection. Third, the proximal lck gene promoter is silenced in single positive T cells and, consequently, is nonfunctional in all T cells that exit the thymus (26). Inasmuch as we had evidence that inactive forms of CaMKIV seemed to be unstable when expressed in mammalian cells in culture (23), we reasoned that the hCaMKIV might be turned over in peripheral T cells. If so, then we could determine whether any effects on T cell activation due to the presence of the mutant hCaMKIV would be reversible. As shown by our results, the vector was well chosen.

T cells that contain a 3-fold excess of mutant to endogenous CaMKIV fail to phosphorylate CREB in response to activating stimuli. Failure to phosphorylate CREB leads to the inability to produce Fos B mRNA. Fos B has been shown to be involved in activation of the IL-2 promoter (1, 2); thus, IL-2 production is also severely attenuated. Splenic T cells indeed no longer contain the mutant hCaMKIV based on evaluation of Western blots. These cells regain the ability to respond to activating stimuli by increasing CREB phosphorylation. They also produce normal levels of IL-2 (data not shown). These data suggest that the mutant hCaMKIV is acting in a dominant/negative manner although we cannot be certain as to the molecular mechanism responsible for the dominant/negative effect. As even higher levels of calspermin are without phenotypic consequence, it is unlikely that the mutant hCaMKIV is nonspecifically sequestering Ca2+/calmodulin that might be needed for other events required for T cell activation. One such event is known to be the activation of calcineurin (5). CaMKIV is found in the nucleus (18), so it is possible that the inactive protein might prevent nuclear entry of CaMKIV or competitively inhibit its ability to interact with other nuclear proteins (including, but not restricted to, substrates such as CREB). Finally, CaMKIV must be phosphorylated by a CaM kinase kinase to become activated (23, 28, 32, 38, 39, 40, 41, 42, 43, 44). Perhaps this reaction is stoichiometric and thus the presence of an excess of an inactive form of CaMKIV prevents activation of the endogenous enzyme by the relevant CaMKIV kinase. Regardless of the mechanism involved, our data implicate CaMKIV as the physiologically relevant CREB kinase in T cells. Inasmuch as a number of other enzymes present in T cells are capable of phosphorylating CREB, this is a remarkable finding. The physiological relevance of phosphorylation of CREB by CaMKIV has also been demonstrated by Bito et al. (24), who used an antisense RNA approach to study expression of immediate early genes in response to electrical stimulation of hippocampal neurons. They also concluded that the important Ca2+-dependent enzyme in this system was CaMKIV.

Barton et al. (15) produced mice that expressed a mutant form of CREB (Ser-133 to Ala) specifically in T cells using the CD2 promoter. Two phenotypic consequences of the presence of the nonphosphorylatable form of CREB are similar to those we find in the mice that express the mutant hCaMKIV. First, the distribution of cells in the thymus is normal in both lines of transgenic mice. Second, both types of cells fail to induce the expression of Fos-B mRNA or produce IL-2 in response to a variety of activating signals. It seems likely that both CaMKIV and CREB are involved in the pathways responsible for activation. However, differences also exist in the phenotypes that result from the expression of the mutant forms of CaMKIV and CREB. Even though the mutant CREB-containing cells cannot produce IL-2, they do up-regulate the expression of the {alpha}-chain of the IL-2 receptor, CD25, in a manner similar to control T cells (15). As shown in Fig. 6BGo, this is not the case for cells containing mutant hCaMKIV, which do increase CD25 expression, but only to about 30% of normal. This indicates that initial cell-surface signaling events are impaired and implicate another role for CaMKIV in T cell activation in addition to its function as a CREB kinase. Perhaps the most obvious difference between the two types of transgenic mice is the degree of thymic cellularity. Although the number of T cells is normal in mice containing mutant CREB, expression of mutant hCaMKIV leads to a 20% reduction compared to control animals. As mentioned above, although the cellularity is reduced, the distribution between CD4-CD8- (double negative, DN), CD4+CD8+ (double positive, DP), CD4+ single positive (SP), or CD8+ SP cells is not disturbed. Also cells expressing mutant hCaMKIV exhibit a markedly decreased rate of survival when placed in culture and incubated under conditions that do not favor activation, whereas cells expressing mutant CREB do not differ from control cells in this assay. The defect that results in reduced thymic cellularity and decreased rate of survival in culture might be linked. An interesting avenue for future investigation is to determine the mechanism responsible for this defect.

Krebs et al. (45) have reported that CaMKIV does not exist in rat brain or thymus at embryonic day 15 (E15) but the mRNA and protein are found 1 day later at E16. At least in culture, the timing of this expression can be advanced by addition of thyroid hormone to the culture medium. The majority of cells in the thymus at E15 are DN (46, 47). Since DP cells begin to accumulate at E16 and this corresponds to the presence of CaMKIV, it is tempting to speculate that the CaMKIV gene is activated as one of the events required to produce DP from DN cells. Hanissian et al. (17) have shown that both DP and SP T cells contain CaMKIV. Moreover the highest concentration is present in DP cells. However, since the murine proximal lck promoter is functional in DN cells both during embryogenesis and in adult thymus (26), the mutant hCaMKIV is also likely to be present in these cells. If this is the case, perhaps it prevents the effects of CaMKIV that could be involved in generation of DP cells. This scenario could result in slowing the conversion of DN to DP cells, which could in turn lead to an increased rate of apoptosis of this population. A general decrease in the number of DN cells would lead to a decrease in the number of DP cells, which would in turn reduce production of SP cells. A sequence of events such as this is one of the few that we can envision that would lead to a decrease in cellularity but no change in the distribution of the remaining T cells, based on the CD4 and CD8 cell surface markers. Alternatively, it is possible that the presence of the mutant hCaMKIV in DN cells might result in a decreased rate of proliferation, and this effect might be independent of a normal physiological role of CaMKIV. Certainly a decreased proliferative capacity of DN cells could also result in decreased cellularity without altering the maturation of the remaining T cells.

Regardless of the mechanism involved, the currently available data would support a role for CaMKIV in DP cells that is independent of its role in the activation of SP cells. Resting DN T cells from adult mice do not contain T cell receptors or normally produce any cytokine. However, cytokine production can be induced by treatment with phorbol ester and ionophore (47). Inasmuch as these cells do not contain CaMKIV, the Ca2+ signal must not involve this enzyme. Even if CaMKII is present in DN cells, activation of this enzyme inhibits cytokine production (10), presumably because it phosphorylates two sites on CREB, and the second site (SER-142) is both dominant and inhibitory (19). Of the other pathways known to be responsive to phorbol ester and ionophore, protein kinase C does not phosphorylate CREB on Ser-133. However, this combination of agents does stimulate signaling through the MAP kinase cascade, and this cascade has been suggested to be Ca2+ sensitive because it is impacted by the CaMKIV pathway. Enslen et al. (44) show that the activation of the CaMKIV cascade can, in turn, activate the MAP kinases ERK-2, JNK-1, and p38 in NG108 cells. This pathway leads to the transcription of Elk-1, c-jun, and ATF-2. Chen et al. (48) have generated mice deficient in c-jun and find that there is a marked decrease in thymic cellularity, a phenotype in common with the mice that express mutant hCaMKIV. Finally, The MAP kinase pathway has been shown to be involved in the differentiation from DN to DP T cells (49). Perhaps, the presence of mutant hCaMKIV prevents the activation of the MAP kinase pathway in DN cells even though CaMKIV is not normally a component of these cells. One could envision that binding of mutant hCaMKIV to a component of this cascade could prevent its activation by another mechanism. This could result in a dominant/negative effect that would be independent of the activation of CREB by CaMKIV. At any rate, many of these suggestions may be tested experimentally, and the availability of transgenic mice expressing mutant hCaMKIV should provide a very useful model system for future research.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
PMA and ionomycin were obtained from Calbiochem (La Jolla, CA). [125I]Protein-A, [{alpha}-32P]deoxy-CTP, and enhanced chemiluminescence (ECL) reagents were purchased from Amersham (Arlington Heights, IL). Phycoerythrocin and fluorescein isothiocyanate-conjugated antibodies for the murine CD4, CD8, and CD25 complexes as well as all tissue culture reagents came from GIBCO (Grand Island, NY). Anti-calspermin antibodies were produced in rabbits housed on site at the Duke University vivarium and had characteristics similar to the antibodies described by Ono et al. (29). A bacterially expressed form of the rat calspermin protein was used as the antigen. We also obtained two independently derived anti-CaMKIV polyclonal antibodies: one was a gift from Talal Chatila (Washington University, St. Louis, MO); the other was generated commercially by Immuno-Dynamics Incorporated (La Jolla, CA). Both antibodies were produced in rabbits against identical synthetic peptides that consisted of the C-terminal 17 amino acids of the human form of CaMKIV. Anti-CREB and anti-phospho-CREB antibodies were obtained from Upstate Biotechnology Inc (Lake Placid, NY). Enzyme-linked immunosorbent assay (ELISA) reagents for the measurement of murine IL-2 production as well as the anti-{alpha}CD3 monoclonal antibody (145.2C11) were provided by Pharmingen (San Diego, CA). All other reagents were of molecular biology grade unless otherwise specified

Transgene Construction and Generation of Mice
Targeted expression of the transgenes to murine thymic T cells was accomplished by utilizing the shuttle vector p1017 [a gift from R. Perlmutter, University of Washington, Seattle] (25). This vector contains the sequence for the mouse proximal lck promoter plus additional sequence derived from the human GH gene (hGH) and has been shown to initiate high level expression of cDNAs specifically in thymic T cells of transgenic mice (27). A 1.5-kb cDNA for the human form of Ca2+/calmodulin-dependent protein kinase type IV (hCaMKIV) that had been mutated to result in a catalytically inactive protein (23) and a 0.5-kb cDNA for the rat form of calspermin (29) were isolated from plasmids by the appropriate restriction digests and subcloned separately into a BamHI site between the lck promoter and the hGH sequences of the p1017 vector as represented diagrammatically in Fig. 1AGo. After confirming that the cDNAs were in the correct orientation by DNA sequencing, the transgenes were linearized and isolated by restriction digestion with NotI (Boehringer Mannheim, Indiana, IN) followed by electrophoresis in a 1% agarose gel (FMC Corp., Rockland, ME). Both transgenes were purified from the agarose using Prep-a-Gene (Bio-Rad, Richmond, CA), aliquoted in 10 mM Tris and 0.1 mM EDTA buffer and stored at -20 C for subsequent microinjection.

B6SLF1/J mice (Jackson Laboratories, Bar Harbor, ME) were used to generate the transgenic mice, because they produce excellent yields of viable eggs for microinjection (C. Bock, personal communication). Ovulation of female mice, male stud service, collection of eggs, microinjection of DNA, and reimplantation of fertilized eggs were done following standard methods previously described (50) and was performed by the Duke Comprehensive Cancer Center Transgenic Core Facility. Mice that expressed the correct DNA fragment as determined by Southern blot analysis were then bred to nontransgenic B6SLF1/J mice to establish viable lines for analysis. All mice were housed at the Duke Cancer Center Isolation Facility under a 12-h light, 12-h dark cycle. Food and water were provided ad libitum, and all care given to the mice was in compliance with the NIH guidelines for the care and use of laboratory animals.

DNA and RNA Analysis
Genotyping of founder mice and subsequent generations of mice was done by Southern blot analysis as previously described (51). In this procedure, 10 µg genomic DNA were digested with SpeI (Boehringer Mannheim) for 12 h at 37 C electrophoresed through a 1% agarose gel and blotted onto Zeta-Probe membranes (Bio-Rad, Richmond, CA). 32P randomly labeled probes were used to confirm the presence of the transgene and to ascertain copy number. For the lck-hCaMKIVkin(-) mice a 500 bp BamHI-BglII fragment derived from the hCaMKIV cDNA 3'-end was used as the probe. In the case of the lck-calspermin transgene, we used the entire 522-bp cDNA encoding rat calspermin. It should be noted that the SpeI restriction site used for Southern analysis was engineered into the p1017 plasmid at both the extreme 5'- and 3'-ends (25, 27) and does not exist in the wild type locus for either CaMKIV or calspermin. Hence, while the cDNA probes also hybridized to the wild type locus, only a single band was detected in Southern blots because the wild type band was substantially larger than the transgene. Total RNA was isolated from tissue after extraction with Utraspec total RNA isolation reagent (Biotex Laboratories, Houston, TX). RNA was subjected to electrophoresis through 1.5% agarose-formaldehyde gels (5 µg/lane) and blotted onto Zeta-Probe membranes (Bio-Rad, Richmond, CA). RNA blots were then hybridized to a 32P randomly labeled cDNA fragment corresponding to the appropriate mRNA. The Fos B probe was made from a l.6-kb HindII fragment of murine Fos B. The GAPDH probe was from a 780-bp PstI/XbaI fragment of human GAPDH. Hybridization was carried out in 500 mM sodium phosphate (pH 7.2), 7% SDS, and 1 mM EDTA at 65 C for 18 h. Washes were done at 65 C in 40 mM sodium phosphate and 1% SDS until background level radioactivity was undetectable. Visualization of blots was accomplished by exposure of the blot to Kodak XAR film (Eastman Kodak, Rochester, NY) at -70 C for up to 24 h.

Primary Cell Culture
Primary cultures of thymic or splenic T cells were established from mice between the ages of 21 and 30 days of age for all experiments. Mice were killed by cervical dislocation, and the thymus or the spleen was harvested. Cells were teased from the organs by placing the organs between two pieces of 40xx nytex nylon mesh (Tetko, Kansas City, MO) submerged in RPMI-1640 (GIBCO) supplemented with 10% FBS, 0.5% penicillin, 0.5% streptomycin, 1x nonessential amino acids, and 0.001 M 2-mercaptoethanol at 4 C and gently pushing down on top of the mesh to rupture the organs. The cells were collected from the supernatant fluid, and the remaining tissue was removed and discarded. This procedure resulted in a T cell purity of >95% from the thymus as assessed by flow cytometry. Splenic T cells were differentiated from B cells based upon the size differential when counting by trypan blue exclusion compared with T cells present in the thymus. This method yielded consistent results, and flow cytometry was employed to confirm the presence of equal numbers of T cells. After two successive washes in RPMI-1640 media, the cells were counted on a hemocytometer by trypan blue exclusion to determine both the number of cells as well as the fraction representing live cells before aliquoting them for experiments.

Survival assays were carried out on aliquots of cells from spleen or thymus. Cells were collected by centrifugation at 1000 rpm for 5 min at 4 C and resuspended in DMEM (GIBCO) supplemented with 10% FBS, 0.5% penicillin, and 0.5% streptomycin. Cells were then recounted on a hemocytometer by trypan blue exclusion, and 4 x 106 live cells were plated in pentuplicate in 24-well Falcon brand plates (Becton Dickinson, San Jose, CA) containing 2 ml DMEM. The cells were then incubated at 37 C in 95% O2/5% CO2. After 30 h the cells were harvested and counted on a hemocytometer by trypan blue exclusion to determine the rate of survival.

Fluorescence-Activated Cell Sorter (FACS) Analysis
All FACS analyses were carried out at the Duke Comprehensive Cancer Center Flow Cytometry Core Facility. Experiments were analyzed within 24 h after completion using a Becton-Dickinson model FACScan with data analysis done using the Cell Quest program (Becton-Dickinson).

CD4/CD8 determinations for thymic and splenic T cells were carried out as previously described (52). Briefly, 1 x 106 T cells were placed into 10 x 75 mm tubes (Becton Dickinson) on ice. The cells were washed two times with PBS containing 5% FBS (FACS buffer). The supernatant fluid was aspirated and 100 µl FACS buffer were added containing either PE-conjugated anti-CD4 (diluted 1:50) or FITC-conjugated anti-CD8 (diluted 1:50) or both phycoerythrin (PE)-CD4 and FITC-CD8. The reaction was allowed to proceed 20 min at 4 C before the reaction was terminated by the addition of fresh FACS buffer. Cells were then briefly washed with fresh FACS buffer and pelleted at 1000 rpm two times. The cells were then fixed with PBS containing 1% formaldehyde.

Quantification of the murine IL-2 receptor on T cells was accomplished by taking an aliquot of 4 x 106 live cells, dividing them in half, and plating the paired samples containing 2 x 106 live cells each into 24- well Falcon Brand plates (Becton-Dickinson) containing fully supplemented RPMI-1640. The final volume of each well was then adjusted to 2 ml. One half of each paired cell sample was left unstimulated while the other half was stimulated with PMA (10 ng/ml) and ionomycin (0.5 µg/ml). After 30 h the cells were harvested, washed in FACS buffer as described above, and immunostained for FACS analysis using FITC-conjugated anti-CD25 antibody (GIBCO) which recognizes the IL-2 receptor {alpha}-chain.

Protein and Cytokine Analysis
Detection of proteins on Western blot was accomplished by either standard radioactivity methods or by chemiluminescence. Expression of protein from the transgene was examined by first extracting protein from 5 x 106 T cells isolated from the thymus or spleen in Laemmli buffer (53). These protein samples were then heated to 95 C, loaded onto a 10% polyacrylamide SDS-containing gel, and run at 100 V for 2 h or until the dye front reached the bottom. The protein was transferred electrophoretically to Immobilon Membranes (Millipore, Bedford, MA). Membranes were blocked for 1 h at room temperature using Tris-buffered saline (TBS) with 5% dried milk and 0.01% Tween 20. Application of the primary antibody for either hCaMKIV or calspermin was then carried out for 2 h at room temperature in TBS followed by six washes in TBS containing 0.01% Tween 20 (TBSt) lasting 10 min each; 1 x 106 cpm/ml of [125I] protein-A was then applied for 1 h in TBS containing 5% dried milk and 0.01% Tween 20 followed by six washes in TBSt for 10 min each. Blots were exposed on Kodak XAR film overnight for visualization. CREB and phospho-CREB detection was accomplished by stimulating 5 x 106 cells with PMA (10 ng/ml) and ionomycin (0.5 µg/ml) or anti-{alpha}-CD3 monoclonal antibody (16 µg/ml) that had been immobilized to 24-well tissue culture plates at 37 C in 95% O2/5% CO2. At various time points, T cells were harvested and lysed in Laemmli buffer as described previously. Proteins were then fractionated by SDS-PAGE on a 10% gel and subjected to Western blotting as described above using commercial antibodies from Upstate Biotechnology Inc. ECL chemiluminescent detection (Amersham) was substituted for [125I] protein-A following the established manufacturers protocols. Blots were quantified by scanning densitometry on a 445 SI PhosphorImager equipped with a personal densitomiter SI (Molecular Dynamics, Sunnyvale, CA). Digital analysis of the scans was completed using the program Image Quant v1.1 (Molecular Dynamics).

To measure production of IL-2, 2 x 105 T cells were placed into a flat bottom 96-well plate (Corning-Costar, Cambridge, MA) containing the RPMI-1640 media previously described. Well volumes were then adjusted to 250 µl, and the cells were stimulated at 37 C in a humidified chamber with PMA (10 ng/ml) and ionomycin (0.5 µg/ml) or 16 µg/ml of the anti-{alpha}-CD3 monoclonal antibody that had been immobilized onto the plate. After 48 h the media were removed for analysis. ELISA measurements were done following the standard protocol distributed with the ELISA components (Pharmingen, San Diego, CA) for measurement of IL-2. Plates were then quantified on a Multiskan MS 96-well plate reader (Lab Systems, Needham Heights, MA)

Statistical Analysis
Initial statistics were performed by a two-way ANOVA utilizing a 95% confidence level. Post-hoc analysis was completed using either the Student-Newman-Keuls test when analyzing data from all three types of mice or a two-tailed Student’s t test for data derived from just two types of mice. All data are presented as the mean ± SD. A level of significance was accepted when P < 0.05


    ACKNOWLEDGMENTS
 
We are very grateful to our colleagues Chris Kane, Libby MacDougall, and Sara Hook for helpful discussions. We also thank Roger Perlmutter (The University of Washington, Seattle, WA) for the targeting vector p1017, Talal Chatila (Washington University, St. Louis, MO) for the antibody to human CaMKIV, and Jeffrey Leiden (University of Chicago, Chicago, IL) for the Fos B probe. We are most appreciative to Cheryl Bock and Wendy Callahan of the Duke Comprehensive Cancer Center Core Transgenic Facility for generation of the transgenic mice and to Mike Cook of the Cancer Center Core Flow Cytometry Facility for performing the FACS analyses.


    FOOTNOTES
 
Address requests for reprints to: Dr. Anthony R. Means, Department of Pharmacology, Box 3813, Duke University Medical Center, Durham, North Carolina 27710.

1 These authors contributed equally to this work. Back

This work was supported by NIH Grant HD-07503 (to A.R.M.). K.A.A. is recipient of a National Research Service Award from the NIH, and M.I. is recipient of a fellowship from Federico II University, Naples, Italy.

Received for publication February 27, 1997. Revision received March 18, 1997. Accepted for publication March 19, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Crabtree GR, Clipstone N 1994 Signal transmission between the plasma membrane and the nucleus of T lymphocytes. Annu Rev Biochem 63:1045–1076[CrossRef][Medline]
  2. Northrop JP, Ho SN, Chen L, Thomas KF, Nolan GP, Admon A, Crabtree GR 1994 NF-AT components define a family of transcription factors targeted in T-cell activation. Nature 369:497–502[CrossRef][Medline]
  3. Jain J, McCaffrey PG, Valge-Archer VE, Rao A 1992 Nuclear factor of activated T cells contains Fos and Jun. Nature 356:801–804[CrossRef][Medline]
  4. Castigli E, Chatila TA, Geha RS 1993 A protein of the AP-1 family is a component of the nuclear factor of activated T lymphocytes. J Immunol 150:3284–3290[Abstract/Free Full Text]
  5. Clipstone NA, Crabtree GR 1992 Identification of calcineurin as a key signaling enzyme in T lymphocyte activation. Nature 357:695–697[CrossRef][Medline]
  6. Ruff VA, Leach KL 1995 Direct demonstration of NFATp dephosphorylation and nuclear localization in activated HT-2 cells using a specific NFATp polyclonal antibody. J Biol Chem 270:22602–22607[Abstract/Free Full Text]
  7. Luo C, Shaw KT, Raghavan A, Aramburu J, Garcia-Cozar F, Perrino BA, Hogan PG, Rao A 1996 Interaction of calcineurin with a domain of the transcription factor NFAT1 that controls nuclear import. Proc Natl Acad Sci USA 93:8907–8912[Abstract/Free Full Text]
  8. Shaw KT, Ho AM, Raghavan A, Kim J, Jain J, Park J, Sharma S, Rao A, Hogan PG 1995 Immunosuppressive drugs prevent a rapid dephosphorylation of transcription factor NFAT1 in stimulated immune cells. Proc Natl Acad Sci USA 92:11205–11209[Abstract]
  9. Schwartz RH 1990 A cell culture model for T lymphocyte clonal anergy. Science 248:1349–1356[Medline]
  10. Nghiem P, Ollick T, Gardner P, Schulman H 1994 Interleukin-2 transcriptional block by the multifunctional Ca2+/calmodulin kinase. Nature 371:347–350[CrossRef][Medline]
  11. Hama N, Paliogianni F, Fessler BJ, Boumpas DT 1995 Calcium/calmodulin-dependent protein kinase II downregulates both calcineurin and protein kinase C-mediated pathways for cytokine gene expression. J Exp Med 181:1217–22[Abstract]
  12. Ullman KS, Northrop JP, Admon A, Crabtree GR 1993 Jun family members are controlled by a calcium-regulated, cyclosporin A-sensitive signaling pathway in activated T lymphocytes. Genes Dev 7:188–196[Abstract]
  13. Sheng M, Greenberg ME 1990 The regulation and function of c-fos and other immediate early genes in the nervous system. Neuron 4:477–485[Medline]
  14. Gonzalez GA, Montminy MR 1989 Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell 59:675–680[Medline]
  15. Barton K, Muthusamy N, Chanyangam M, Fischer C, Clendenin C, Leiden JM 1996 Defective thymocyte proliferation and IL-2 production in transgenic mice expressing a dominant-negative form of CREB. Nature 379:81–85[CrossRef][Medline]
  16. Frangakis MV, Chatila T, Wood ER, Sahyoun N 1991 Expression of a neuronal Ca2+/calmodulin-dependent protein kinase, CaM kinase-Gr, in rat thymus. J Biol Chem 266:17592–17596[Abstract/Free Full Text]
  17. Hanissian SH, Frangakis M, Bland MM, Jawahar S, Chatila TA 1993 Expression of a Ca2+/calmodulin dependent protein kinase, CaM kinase-Gr, in human T lymphocytes. Regulation of kinase activity by T cell receptor signaling. J Biol Chem 268:20055–20063[Abstract/Free Full Text]
  18. Jensen KF, Ohmstede CA, Fisher RS, Sahyoun N 1991 Nuclear and axonal localization of Ca2+/calmodulin-dependent protein kinase type Gr in rat cerebellar cortex. Proc Natl Acad Sci USA 88:3850–2853
  19. Sun P, Enslen H, Myung PS,Maurer RA 1994 Differential activation of CREB by Ca2+/calmodulin-dependent protein kinases type II and type IV involves phosphorylation of a site that negatively regulates activity. Genes Dev 8:2527–2539[Abstract]
  20. Enslen H, Sun P, Brickey D, Soderling SH, Klamo E, Soderling TR 1994 Characterization of Ca2+/calmodulin-dependent protein kinase IV; role in transcription regulation. J Biol Chem 269:15520–15527[Abstract/Free Full Text]
  21. Matthews RP, Guthrie CR, Wailes LM, Zhao X, Means AR, McKnight GS 1994 Calcium/calmodulin-dependent protein kinase types II and IV differentially regulate CREB-dependent gene expression. Mol Cell Biol 14:6107–6116[Abstract]
  22. Ho N, Gullberg M, Chatila T 1996 Activation protein 1-dependent transcriptional activation of interleukin 2 gene by Ca2+/calmodulin kinase type IV/Gr. J Exp Med 184:101–112[Abstract]
  23. Chatila T, Anderson KA, Ho N, Means AR 1996 A Unique phosphorylation-dependent mechanism for the activation of Ca2+/calmodulin-dependent protein kinase type IV/Gr. J Biol Chem 271:21542–21548[Abstract/Free Full Text]
  24. Bito H, Deisseroth K, Tsien RW 1996 CREB Phosphorylation and dephosphorylation: a Ca2+-and stimulus duration-dependent switch for hippocampal gene expression. Cell 87:1203–1214[Medline]
  25. Allen JM, Forbush KA, Perlmutter RM 1992 Functional dissection of the lck proximal promoter. Mol Cell Biol 12:2758–2768[Abstract]
  26. Reynolds PJ, Lesley J, Trotter J, Schulte R, Hyman R, Sefton BM 1990 Changes in the relative abundance of type I and type II lck mRNA transcripts suggest differential promoter usage during T-cell development. Mol Cell Biol 10:4266–4270[Medline]
  27. Chaffin KE, Beals CR, Wilkie TM, Forbush KA, Simon MI, Perlmutter RM 1990 Dissection of thymocyte signaling pathways by in vivo expression of pertussis toxin ADP-ribosyltransferase. EMBO J 9:3821–3829[Abstract]
  28. Selbert MA, Anderson KA, Huang QH, Goldstein EG, Means AR, Edelman AM 1995 Phosphorylation and activation of Ca2+-calmodulin-dependent protein kinase IV by Ca2+-calmodulin-dependent protein kinase Ia kinase; phosphorylation of threonine 196 is essential for activation. J Biol Chem 270:17616–17621[Abstract/Free Full Text]
  29. Ono T, Slaughter GR, Cook RG, Means AR 1989 Molecular cloning, sequence and distribution of rat calspermin, a high affinity calmodulin-binding protein. J Biol Chem 264:2081–2087[Abstract/Free Full Text]
  30. Means AR, Cruzalegui F, LeMagueresse B, Needleman DS, Slaughter GR, Ono T 1991 A novel Ca2+/calmodulin-dependent protein kinase and a male germ cell-specific calmodulin-binding protein are derived from the same gene. Mol Cell Biol 11:3960–3971[Medline]
  31. Sun Z, Sassone-Corsi P, Means AR 1995 Calspermin gene transcription is regulated by two cyclic AMP response elements contained in an alternative promoter in the calmodulin kinase IV gene. Mol Cell Biol 15:561–571[Abstract]
  32. Park IK, Soderling TR 1995 Activation of Ca2+/calmodulin-dependent protein kinase (CaM-kinase) IV by CaM-kinase kinase in Jurkat T lymphocytes. J Biol Chem 270:30464–30469[Abstract/Free Full Text]
  33. Ginty DD, Kornhauser JM, Thompson MA, Bading H, Mayo KE, Takahashi JS, Greenberg ME 1993 Regulation of CREB phosphorylation in the suprachiasmatic nucleus by light and a circadian clock. Science 260:238–241[Medline]
  34. Sun P, Lou L, Maurer RA 1996 Regulation of activating transcription factor-1 and the cAMP response element-binding protein by Ca2+/calmodulin-dependent protein kinases type I, II, and IV. J Biol Chem 271:3066–3073[Abstract/Free Full Text]
  35. Carding SR, Hayday AC, Bottomly K 1991 Cytokines in T-cell development. Immunol Today 12:239–245[Medline]
  36. Fischer M, MacNeil I, Suda T, Cupp JE, Shortman K, Zlotnik A 1991 Cytokine production by mature and immature thymocytes. J Immunol 146:3452–3456[Abstract/Free Full Text]
  37. Alberola-lla J, Forbush KA, Seger R, Krebs EG, Perlmutter RM 1995 Selective requirement for MAP kinase activation in thymocyte differentiation. Nature 373:620–623[CrossRef][Medline]
  38. Okuno S, Kitani T, Fujisawa H 1994 Purification and Characterization of Ca2+/calmodulin-dependent protein kinase IV kinase from rat brain. J Biochem 116:923–930[Abstract]
  39. Enslen H, Tokumitsu H, Soderling TR 1995 Phosphorylation of CREB by CaM-kinase IV activated by CaM-kinase IV kinase. Biochem Biophys Res Commun 207:1038–1043[CrossRef][Medline]
  40. Tokumitsu H, Brickey DA, Glod J, Hidaka H, Sikela J, Soderling TR 1994 Activation Mechanisms for Ca2+/calmodulin-dependent protein kinase IV. J Biol Chem 269:28640–28647[Abstract/Free Full Text]
  41. Tokumitsu H, Enslen H, Soderling TR 1995 Characterization of a Ca2+/calmodulin-dependent protein kinase cascade; molecular cloning and expression of calcium/calmodulin-dependent protein kinase kinase. J Biol Chem 270:19320–19324[Abstract/Free Full Text]
  42. Tokumitsu H, Soderling TR 1996 Requirements for calcium and calmodulin in the calmodulin kinase activation cascade. J Biol Chem 271:5617–5622[Abstract/Free Full Text]
  43. Edelman AM, Mitchelhill KI, Selbert MA, Anderson KA, Hook SS, Stapleton D, Goldstein EG, Means AR, Kemp BE 1996 Multiple Ca2+-calmodulin-dependent protein kinase kinases from rat brain; purification, regulation by Ca2+-calmodulin, and partial amino acid sequence. J Biol Chem 271:10806–10810[Abstract/Free Full Text]
  44. Enslen H, Tokumitsu H, Stork PJS, Davis RJ, Soderling TR 1996 Regulation of mitogen-activated protein kinases by a calcium/calmodulin-dependent protein kinase cascade. Proc Natl Acad Sci USA 93:10803–10808[Abstract/Free Full Text]
  45. Krebs J, Means RL, Honegger P 1996 Induction of calmodulin kinase IV by the thyroid hormone during the development of rat brain. J Biol Chem 271:11055–11058[Abstract/Free Full Text]
  46. Montgomery RA, Dallman MJ 1991 Analysis of cytokine gene expression during fetal thymic ontogeny using the polymerase chain reaction. J Immunol 147:554–560[Abstract/Free Full Text]
  47. Zuniga-Pflucker JC, Schwartz HL, Lenardo MJ 1993 Gene transcription in differentiating immature T cell receptorneg thymocytes resembles antigen-activated mature T cell. J Exp Med 178:1139–1149[Abstract]
  48. Chen J, Stewart V, Spyrou G, Hilberg F, Wagner EF, Alt FW 1994 Generation of normal T and B lymphocytes by c-jun deficient embryonic stem cells. Immunity 1:65–72[Medline]
  49. Crompton T, Gilmour KC, Owen MJ 1996 The MAP kinase pathway controls differentiation from double-negative to double-positive thymocyte. Cell 86:243–251[Medline]
  50. Hogan B, Beddington R, Costantini F, Lacy E 1994 Manipulating the Mouse Embryo—A Laboratory Manual, ed. 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  51. Ribar TJ, Epstein PN, Overbeek PA, Means AR 1995 Targeted overexpression of an inactive calmodulin that binds Ca2+ to the mouse pancreatic B-cell results in impaired secretion and chronic hyperglycemia. Endocrinology 136:106–115[Abstract]
  52. Garvin AM, Abraham KM, Forbush KA, Farr AG, Davison BL, Perlmutter RM 1990 Disruption of thymocyte development and lymphogenesis induced by SV40 T-antigen. Int Immunol 2:173–180[Medline]
  53. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage t4. Nature 227:680–685[Medline]