Donald A. Withers

Sphingosine-dependent Protein Kinase-1, Directed to 14-3-3, Is Identified as the Kinase Domain of Protein Kinase Cδ

Some protein kinases are known to be activated by d-erythro-sphingosine (Sph) or N,N-dimethyl-d-erythro-sphingosine (DMS), but not by ceramide, Sph-1-P, other sphingolipids, or phospholipids. Among these, a specific protein kinase that phosphorylates Ser60, Ser59, or Ser58 of 14-3-3β, 14-3-3η, or 14-3-3ζ, respectively, was termed “sphingosine-dependent protein kinase-1” (SDK1) (Megidish, T., Cooper, J., Zhang, L., Fu, H., and Hakomori, S. (1998) J. Biol. Chem. 273, 21834–21845). We have now identified SDK1 as a protein having the C-terminal half kinase domain of protein kinase Cδ (PKCδ) based on the following observations. (i) Large-scale preparation and purification of proteins showing SDK1 activity from rat liver (by six steps of chromatography) gave a final fraction with an enhanced level of an ∼40-kDa protein band. This fraction had SDK1 activity ∼50,000-fold higher than that in the initial extract. (ii) This protein had ∼53% sequence identity to the Ser/Thr kinase domain of PKCδ based on peptide mapping using liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry data. (iii) A search for amino acid homology based on the BLAST algorithm indicated that the only protein with high homology to the ∼40-kDa band is the kinase domain of PKCδ. The kinase activity of PKCδ did not depend on Sph or DMS; rather, it was inhibited by these sphingoid bases, i.e. PKCδ did not display any SDK1 activity. However, strong SDK1 activity became detectable when PKCδ was incubated with caspase-3, which releases the ∼40-kDa kinase domain. PKCδ and SDK1 showed different lipid requirements and substrate specificity, although both kinase activities were inhibited by common PKC inhibitors. The high susceptibility of SDK1 to Sph and DMS accounts for their important modulatory role in signal transduction.

Deletion of A-antigen in a human cancer cell line is associated with reduced promoter activity of CBF/NF-Y binding region, and possibly with enhanced DNA methylation of A transferase promoter.

Employing blood group A− and A+ clones derived from the same parental colonic cancer cell lines, we studied the molecular mechanism of deletion/ reduction vs. continuous expression of A antigen in A tumors, a crucial determinant of human tumor malignancy. A− transferase mRNA level in one of the A− clones (A− SW480) was undetectable, while that in A+ SW480 was strongly detectable by semiquantitative RT-PCR. Relatively lower (∼1/3) transcript level was detectable in another A− clone (A− HT29) in comparison to A+ HT29 by the same RT-PCR procedure, although none of these tumor cell lines showed detectable level of A transcript by Northern blotting or RNase protection methods. Therefore, subsequent studies were performed employing A− vs. A+ SW480 clones. Deletion of A transcript in A− cells was not due to gene deletion, since Southern blot analysis showed equal presence of genomic DNA regardless of A− vs. A+ (SW480 or HT29) or B+ (KATOIII) tumor cells. Two transcriptional control mechanisms leading to differences of A expression in SW480 cells are indicated.i. Luciferase assay in A− and A+ SW480 cells showed that promoter activities of segments of 5′ flanking sequence of ABO gene reflected transcript levels in these cell lines. The enhancing activity of a 43bp tandem repeat unit located between −3899 to −3618 was reduced in A− compared to A+ cells.ii. Distinct differences in the pattern of CpG dinucleotide methylation were found in A− vs. A+ cells. Therefore, the methylation process of A promoter DNA may be another important factor controlling A activity in SW480 tumor cells.Since proliferation and motility of tumor cells are associated closely with A expression, transcription control mechanism for expression of A transferase as described above may be of crucial importance in defining human tumor malignancy.

Ganglioside DSGb5, preferred ligand for Siglec-7, inhibits NK cell cytotoxicity against renal cell carcinoma cells

In renal cell carcinoma (RCC), the presence of higher gangliosides correlates with systematic metastasis. Disialosyl globopentaosylceramide (DSGb5) was identified previously as one of the major gangliosides from RCC tissues. Siglec-7 (sialic acid-binding Ig-like lectin-7), expressed on natural killer (NK) cells as an inhibitory receptor, has a striking preference for internally branched α2,6-linked disialic gangliosides such as DSGb5. To clarify the functional role of DSGb5 in RCC metastases, we have investigated whether DSGb5 expressed on RCC cells can modulate NK cell cytotoxicity in a Siglec-7-dependent manner. The binding activity of RCC cells to Siglec-7-Fc fusion protein was specifically inhibited by anti-DSGb5 monoclonal antibody and transfection of siRNA for ST6GalNAcVI (synthetase of DSGb5). These observations showed that Siglec-7-Fc fusion protein specifically bound to DSGb5 expressed on RCC cells. In contrast, the sialic acid-binding site of Siglec-7 on NK cells was masked by cis interactions with endogenous sialoconjugates at the cell surface, but it could be unmasked by sialidase treatment of the NK cells. Following sialidase treatment of NK cells, NK cell cytotoxicity against RCC cells with high DSGb5 expression was significantly decreased relative to cells with low DSGb5 expression. These findings indicate that such NK cell cytotoxicity against RCC cells could be inhibited by the interaction between Siglec-7 on effecter cells and DSGb5 on target cells. The results of the present study suggest that DSGb5 expressed on RCC cells can downregulate NK cell cytotoxicity in a DSGb5-Siglec-7-dependent manner and that RCC cells with DSGb5 create favorable circumstance for their own survival and metastases.

Erratum to: Binding specificity of siglec7 to disialogangliosides of renal cell carcinoma: possible role of disialogangliosides in tumor progression (FEBS 24901): [FEBS Letters 498 (2001) 116–120]

Erratum to: Binding speci¢city of siglec7 to disialogangliosides of renal cell carcinoma: possible role of disialogangliosides in tumor progression (FEBS 24901) [FEBS Letters 498 (2001) 116^120]C Akihiro Itoa;b;c;*, Kazuko Handaa;b, Donald A. Withersa;b, Makoto Satohc, Sen-itiroh Hakomoria;b aPaci¢c Northwest Research Institute, 720 Broadway, Seattle, WA 98122-4327, USA bDepartments of Pathobiology and Microbiology, University of Washington, Seattle, WA 98195, USA cDepartment of Urology, Tohoku University School of Medicine, Sendai, Japan