ABSTRACT
The outcome of thymocyte selection is influenced by the nature of Ca2+ signals transduced by the TCR. Robust Ca2+ responses characterize high-affinity, negatively selecting peptide/TCR interactions, while modest responses typify lower-affinity, positively selecting interactions. To elucidate mechanisms by which thymocytes decode distinct Ca2+ signals, we examined selection events in mice lacking Ca2+/calmodulin-dependent protein kinase type IV/Gr (CaMKIV/Gr), which is enriched in thymocytes. CaMKIV/Gr-deficient thymocytes exhibited impaired positive selection and defective Ca2+-dependent gene transcription. Significantly, CaMKIV/Gr deficiency raised the selection threshold of peptide/TCR interactions such that a peptide that normally induced weak negative selection instead promoted positive selection. These results demonstrate an important role for CaMKIV/Gr in sensitizing thymocytes to selection by low-affinity peptides.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/physiology , T-Lymphocytes/cytology , T-Lymphocytes/enzymology , Thymus Gland/cytology , Thymus Gland/enzymology , Animals , Breeding , Calcium/physiology , Calcium-Calmodulin-Dependent Protein Kinase Type 4 , Calcium-Calmodulin-Dependent Protein Kinases/deficiency , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Cell Differentiation/genetics , Cell Differentiation/immunology , Crosses, Genetic , Gene Expression Regulation, Developmental/immunology , H-Y Antigen/genetics , Hemoglobins/genetics , Mice , Mice, Inbred AKR , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Receptors, Antigen, T-Cell/genetics , T-Lymphocytes/metabolism , Thymus Gland/metabolismABSTRACT
The calcium/calmodulin-dependent protein kinase type IV/Gr (CaMKIV/Gr) is expressed in male germ cells and spermatids and has been implicated in controlling the differentiation of germ cells into mature spermatozoa. The function of CaMKIV/Gr in spermatogenesis was investigated using CaMKIV/Gr-deficient mice generated by targeted gene disruption. CaMKIV/Gr-deficient males exhibited normal spermatogenesis, and their fertility was similar to that of wild-type littermates. Notwithstanding the function of CaMKIV/Gr as an activator of cAMP response element (CRE)-dependent transcription, mRNA levels of several testis-specific CRE modulator (CREM)-regulated genes were unaltered. These results indicate that CaMKIV/Gr is not essential for spermatogenesis or for CRE-regulated gene transcription in the testis.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/physiology , DNA-Binding Proteins/physiology , Isoenzymes/physiology , Repressor Proteins , Spermatogenesis , Spermatozoa/physiology , Transcription, Genetic , Alternative Splicing , Animals , Blotting, Northern , Calcium-Calmodulin-Dependent Protein Kinases/deficiency , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Crosses, Genetic , Cyclic AMP Response Element Modulator , DNA/analysis , Gene Targeting , In Situ Nick-End Labeling , Isoenzymes/deficiency , Isoenzymes/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Organ Size , Reverse Transcriptase Polymerase Chain Reaction , Testis/anatomy & histologyABSTRACT
X-linked autoimmunity-allergic disregulation syndrome (XLAAD) is an X-linked recessive immunological disorder characterized by multisystem autoimmunity, particularly early-onset type 1 diabetes mellitus, associated with manifestations of severe atopy including eczema, food allergy, and eosinophilic inflammation. Consistent with the allergic phenotype, analysis of two kindreds with XLAAD revealed marked skewing of patient T lymphocytes toward the Th2 phenotype. Using a positional-candidate approach, we have identified in both kindreds mutations in JM2, a gene on Xp11.23 that encodes a fork head domain-containing protein. One point mutation at a splice junction site results in transcripts that encode a truncated protein lacking the fork head homology domain. The other mutation involves an in-frame, 3-bp deletion that is predicted to impair the function of a leucine zipper dimerization domain. Our results point to a critical role for JM2 in self tolerance and Th cell differentiation.
Subject(s)
Autoimmune Diseases/genetics , Diabetes Mellitus, Type 1/genetics , Food Hypersensitivity/genetics , Genetic Linkage/genetics , Nuclear Proteins/genetics , Transcription Factors/genetics , X Chromosome/genetics , Amino Acid Sequence , Autoimmune Diseases/immunology , Base Sequence , Cell Differentiation , DNA Mutational Analysis , Diabetes Mellitus, Type 1/immunology , Female , Food Hypersensitivity/immunology , Forkhead Transcription Factors , Haplotypes , Humans , Leucine Zippers , Male , Molecular Sequence Data , Mutation/genetics , Nuclear Proteins/chemistry , Nuclear Proteins/immunology , Pedigree , Protein Structure, Tertiary , RNA Splice Sites/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Syndrome , Th2 Cells/cytology , Th2 Cells/immunology , Transcription Factors/chemistry , Transcription Factors/immunology , X Chromosome/immunologyABSTRACT
The Ca(2+)/calmodulin-dependent protein kinase type IV/Gr (CaMKIV/Gr) is a key effector of neuronal Ca(2+) signaling; its function was analyzed by targeted gene disruption in mice. CaMKIV/Gr-deficient mice exhibited impaired neuronal cAMP-responsive element binding protein (CREB) phosphorylation and Ca(2+)/CREB-dependent gene expression. They were also deficient in two forms of synaptic plasticity: long-term potentiation (LTP) in hippocampal CA1 neurons and a late phase of long-term depression in cerebellar Purkinje neurons. However, despite impaired LTP and CREB activation, CaMKIV/Gr-deficient mice exhibited no obvious deficits in spatial learning and memory. These results support an important role for CaMKIV/Gr in Ca(2+)-regulated neuronal gene transcription and synaptic plasticity and suggest that the contribution of other signaling pathways may spare spatial memory of CaMKIV/Gr-deficient mice.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cerebral Cortex/physiology , Cyclic AMP Response Element-Binding Protein/metabolism , Hippocampus/physiology , Maze Learning/physiology , Motor Activity/physiology , Neuronal Plasticity/physiology , Neurons/physiology , Synapses/physiology , Animals , Brain/physiology , Calcium Signaling/physiology , Calcium-Calmodulin-Dependent Protein Kinase Type 4 , Calcium-Calmodulin-Dependent Protein Kinases/deficiency , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Electric Stimulation , Long-Term Potentiation , Male , Memory , Mice , Mice, Knockout , Posture , Purkinje Cells/physiology , Pyramidal Cells/physiology , Reverse Transcriptase Polymerase Chain Reaction , SwimmingABSTRACT
Ca(2+) induction of a subset of cellular and viral immediate-early activation genes in lymphocytes has been previously mapped to response elements recognized by the MEF2 family of transcription factors. Here, we demonstrate that Ca(2+) activation of MEF2 response elements in T lymphocytes is mediated in synergy by two Ca(2+)/calmodulin-dependent enzymes, the phosphatase calcineurin, and the kinase type IV/Gr (CaMKIV/Gr), which promote transcription by the MEF2 family members MEF2A and MEF2D. Calcineurin up-regulates the activity of both factors by an NFAT-dependent mechanism, while CaMKIV/Gr selectively and independently activates MEF2D. These results identify MEF2 proteins as effectors of a pathway of gene induction in T lymphocytes which integrates diverse Ca(2+) activation signals and may be broadly operative in several tissues.
