Nucleocytoplasmic trafficking of the Syk protein tyrosine kinase is mediated by an unconventional localization signal and is regulated by protein kinase C

Jianjie Hu, Purdue University

Abstract

The protein-tyrosine kinase, Syk, is an essential component of the B cell antigen receptor (BCR) signaling pathway. To explore the intracellular localization of Syk, we have expressed in Syk-deficient DT40 B cells, a chimeric protein containing green fluorescent protein fused to the C-terminus of Syk. In unstimulated cells, Syk is distributed in both the cytoplasm and nucleus. This observation is further confirmed by our indirect immunofluorescence and cellular fractionation studies. However, no nuclear localization signal has been discovered on Syk. To investigate the molecular mechanism of the nuclear localization of Syk, we have expressed in Syk-deficient DT40 B cells a series of Syk deletion mutants coupled to green fluorescent protein. An 11 amino acid sequence (EVYLDRSLLTL) located at the C-terminal end of the linker B region is found to be required for the nuclear localization of Syk. The subcellular localization of Syk changes dramatically following different stimulations. At long time points following BCR engagement, Syk becomes excluded from the nucleus in a significant proportion of cells. A catalytically inactive Syk-GFP does not support receptor signaling and does not exhibit this nuclear exclusion phenomenon, suggesting that signals must be sent from the BCR to the cell interior to trigger nuclear export. To examine the nature of these signals and the translocation process, we are using pharmacological stimulators and inhibitors of downstream signal transduction pathways. Our studies indicate that this redistribution event is dependent on signals sent from the ligated BCR complex, require the activation of protein kinase C and can be mimicked by treatment of cells with phorbol 12-myristate 13-acetate (PMA). The delayed kinetics of Syk translocation is due to a requirement for new protein synthesis.

Degree

Ph.D.

Advisors

Geahlen, Purdue University.

Subject Area

Molecular biology

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