Long-term potentiation (LTP) is an activity-dependent enhancement of synaptic efficacy, considered a model of learning and memory. The biochemical cascade producing LTP requires activation of Src, which upregulates the function of NMDA receptors (NMDARs), but how Src becomes activated is unknown. Here, we show that the focal adhesion kinase CAKbeta/Pyk2 upregulated NMDAR function by activating Src in CA1 hippocampal neurons. Induction of LTP was prevented by blocking CAKbeta/Pyk2, and administering CAKbeta/Pyk2 intracellularly mimicked and occluded LTP. Tyrosine phosphorylation of CAKbeta/Pyk2 and its association with Src was increased by stimulation that produced LTP. Finally, CAKbeta/Pyk2-stimulated enhancement of synaptic AMPA responses was prevented by blocking NMDARS, chelating intracellular Ca(2+), or blocking Src. Thus, activating CAKbeta/Pyk2 is required for inducing LTP and may depend upon downstream activation of Src to upregulate NMDA receptors.

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ABSTRACT. Cell adhesion kinase β (CAKβ/PYK2) is a protein-tyrosine kinase of the focal adhesion kinase (FAK) family. Whereas FAK predominantly localizes at focal adhesions, CAKβ localizes at the perinuclear region in fibroblasts. Here we expressed in cultured cells two point mutants of CAKβ, P717A and P859A, each of which had lost one of its two PXXP motifs, the ligand sequence for SH3 domains, found at the CAKβ C-terminal region. We observed a remarkable change in the subcellular distribution of the P859A mutant; while that of the P717A mutant was the same as the wild type. The P859A mutant localized exclusively in the cell nucleus in all cell lines examined. Wild-type CAKβ also accumulated in the nucleus when cells were treated with an inhibitor of the nuclear export of proteins. These results indicate that CAKβ shuttles between the cytoplasm and the nucleus. On nuclear accumulation of P859A-CAKβ, a CAKβ-binding protein, Hic-5, also accumulated in the nucleus. P859A-CAKβ and co-expressed Hic-5 formed nuclear speckles, in which one other CAKβ-binding protein, p130Cas , was also concentrated. These findings on nuclear translocation of CAKβ imply that CAKβ may regulate nuclear processes such as transcription, particularly because Hic-5 was recently shown to be a coactivator of nuclear receptors.

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Cell adhesion kinase beta (CAKbeta) is a protein tyrosine kinase closely related to focal adhesion kinase (FAK) in structure. CAKbeta contains two proline-rich sequences within its C-terminal region. Since proline-rich sequences present in the corresponding region of FAK are known to mediate protein-protein interactions by binding to SH3 domains, we investigated binding of CAKbeta to a panel of SH3 domains. Affinity precipitation from rat brain lysate revealed selective interactions of CAKbeta with glutathione S-transferase (GST)-fused SH3 domains of p130(Cas)(Cas)-related proteins and Graf. Mutational analysis indicated that the proline-rich sequences of CAKbeta mediate this interaction. Each of the two proline-rich sequences fused to GST bound directly to these SH3 domains in dot blot analysis. A competitive binding assay revealed that the first proline-rich sequence of CAKbeta preferentially associated with the SH3 domain of Cas. The second proline-rich sequence of CAKbeta bound to the SH3 domain of Graf with higher specificity than the corresponding proline-rich sequence of FAK. Finally, we showed co-immunoprecipitation of CAKbeta with Graf from rat brain lysate. These results indicate that CAKbeta associates in vivo with Graf through its SH3 domain.

We have identified a novel cDNA clone, named AZI, obtained from a cDNA library of mRNA prepared from C3H10T1/2 cells that had been transiently exposed to 5-azacytidine, a potent demethylating reagent. The amount of transcript increased with 5-azacytidine treiltment of C3H10T1/2 cells and the transcript was highly expressed in mouse testis. As the mutant mouse jsdljsd, which has a defect in germ cell maturation, barely expressed the transcript, the message was expected to be expressed specifically in spermatocytes. The mRNA was detected at significant levels in the testes from mice aged 16 days after birth, suggesting that its expression started at the pachytene spermatocyte stage. The elucidated nucleotide sequence contained a 2841 -nucleotide open reading frame, and the expected amino acid sequence had a molecular mass of 107 254 Da. Specific antibodies raised against the fusion protein including glutathione S-transferase revealed an approximately 130-kDa band of a translation product in testis and in cultured cells transfected with AZ1 cDNA in the expression vector on Western-blot analysis. The protein was localized to the pre-acrosome region of round and elongated spermatids. However, it was not detected at a more advanced stage of spermatids, i.e. just before their release from Sertoli cells. This protein may play an important role in spermatogenesis.Keywords: DNA demethylation ; spermatogensis ; acrosome ; cDNA cloning.The expression of mammalian genes is often correlated with the methylation state of the CpG sequences in their promoter regions [l]. The promoter regions of house-keeping genes are undermethylated in all cells. In contrast, tissue-specific genes are usually methylated at the 5 position of cytosine in the CpG sequences, except in the tissues in which they are expressed [ 21. Transient exposure of mouse C3H1 OT1/2 cells to 5-azacytidine, a cytosine analogue that cannot be methylated at the 5 position and which is also a potent inhibitor of DNA methyltransferase [3], causes the cells to differentiate into chondrocytes, adipocytes, and myotubes [4], or to undergo transformation [ S ] . The underlying cause of these changes in phenotype is the induction of the genes regulating the phenotype through undermethylation of the genes, such as the myoDl gene for myotubes 161 and endogenous retroviral genes for transformation IS]. One of the possible important roles of DNA methylation in germ cells is the marking of specific genes to determine if they should be expressed at an early stage of embryogenesis. This is called parental or genomic imprinting [7]. At the same time, many tissuespecific genes should be expressed during oogenesis or sperma- University, 3-2, Yamadaoka, Suita, Osaka, Japan 565 togenesis, as these cells are very specialized. In particular, male germ cells undergo drastic differentiation during spermatogenesis. Recently, many genes have been reported to be transcribed post-meiotically in animal male gametes [8, 91. Among these, the expression of the phosphoglycerate kinase 2 gene and t...

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