The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
Lymphocyte Isoforms of Mouse p50 LSP1, Which Are Phosphorylated in Mitogen-Activated T Cells, Are Formed through Alternative Splicing and Phosphorylation
Naoki MatsumotoKiyoshi KitaSomei KojimaKazuo YamamotoTatsuro IrimuraMasaru MiyagiSusumu TsunasawaSatoshi Toyoshima
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1995 Volume 118 Issue 1 Pages 237-243

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Abstract

p50 is phosphorylated in mitogen-stimulated T cells, and translocated from the membrane to the cytosol after activation of protein kinase C. Sequence analysis of p50 revealed that it is identical with LSP1, a putative calcium-binding and actin-binding protein. The lymphocyte form of p50 exhibits heterogeneity in the apparent molecular mass on SDS-PAGE, 50 and 52 kDa (pp50 and pp52), and each isoform exhibits heterogeneity in the isoelectric point, when examined by two-dimensional PAGE. When the two molecular mass variants of p50 were dephosphorylated with alkaline phosphatase, both isoforms showed the same apparent molecular mass of 50 kDa on SDS-PAGE, but could be distinguished by their distinct isoelectric points. Dephosphorylated pp50 (p50a) has an acidic pI compared with dephosphorylated pp52 (p50b). Comparison of the peptide maps of purified p50a and p50b on HPLC revealed that the difference was limited to one peptide peak. NH2-terminal sequence and mass spectrometric analyses of these peptides showed that the peptides derived from p50a and p50b had the same NH2-terminal amino acid sequence up to eight residues, but had distinct molecular masses, 5, 533.4 and 6, 318.6 Da, respectively. These data suggested that pp52 (p50b) is the product of the previously cloned cDNA and the reduction in the molecular mass of the p50a-derived peptide could be explained by deletion of six amino acid residues, EHLIRH or HLIRHQ. To verify this prediction, we examined the mRNA encoding p50 by means of the reverse transcriptase-polymerase chain reaction and found the presence of an mRNA that had a deletion of 18 bp, that results in a deletion of six amino acid residues, HLIRHQ (158-163), after translation. These data, and the results of transfection and expression of the two types of p50 cDNA in BALB/3T3 fibroblasts indicate that alternative splicing and phosphorylation generate the multiple isoforms of p50 in lymphocytes.

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© The Japanese Biochemical Society
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