Jin Hong Liu

Functional association of TGF-β receptor II with cyclin B

Utilizing the cytoplasmic tail of Transforming Growth Factor Receptor Type II (TGFβ RII) as bait in a yeast two hybrid system, we have identified human cyclin B2 as a direct physical partner of TGFβ RII. Analysis of deletion mutants of glutathione-S-transferase (GST)-cyclin B2 mapped its binding domain for TGFβ RII to the C-terminal and revealed a negative regulatory region immediately upstream of the cyclin box. Using recombinant proteins, Cdc2 was demonstrated to indirectly interact with TGFβ RII via cyclin B2. This interaction was reproduced in THP-1 monocytic cells, where TGFβ treatment markedly enhanced the ability of cyclin B2 and, correspondingly, Cdc2 from TGFβ-treated THP-l cells, to bind the GST-TGFβ RII fusion protein. More importantly, TGFβ RII co-precipitated with cyclin B2 in TGFβ-treated THP-1 cells. TGFβ treatment also caused threonine phosphorylation of Cdc2 in the TGFβ RII-cyclin B2-Cdc2 complex in THP1 cells, in parallel with down regulation of Cdc2 function as measured by histone H1 kinase activity. Cyclin B1 had the same capacity to bind TGFβ RII and mediate indirect Cdc2 binding. These results suggest an alternative mechanism that cell cycle arrest in the G1/S phase caused by TGFβ may, in part, be due to inactivation of cyclin B/Cdc2 kinase, which is needed for entry into the G2/M phase.

IL-2 Induces the Association of IL-2Rο, lyn, and MAP Kinase ERK-1 in Human Neutrophils

IL-2, first identified as a T cell growth factor, has been proven to activate many cell types including polymorphonuclear neutrophils (PMN3). However, the mechanisms involved in PMN activation, especially the signaling pathways used by the IL-2R, are currently unknown. Here we demonstrate that IL-2 has the ability to induce protein tyrosine kinases in human PMN, and we provide the first evidence that lyn kinase is activated and physically associated with MAP kinase/ERK1. Co-immunoprecipitation experiments with anti-IL-2Rbeta and Western blotting with anti-p53/56lym revealed that lyn protein was present in IL-2R precipitates and that the association of lyn with IL-2Rbeta was markedly elevated by IL-2 stimulation. Furthermore the activity of lyn kinase, evaluated by an in vitro kinase assay with enolase as a substrate, increased following IL-2 stimulation. Another important finding was that, upon IL-2 activation, MAPK/ERK1 was also phosphorylated in PMN. A direct association between lyn and ERK1 was initially demonstrated by co-immunoprecipitation/Western blotting and then definitively proven by the use of a GST-ERK1 fusion protein. We showed that ERK1 binds lyn only in IL-2 stimulated PMN, but not in unstimulated PMN. These results suggest that IL-2 can promote the association of lyn protein tyrosine kinase with IL-2Rbeta as well as the direct binding of MAPK/ERK1 to lyn. The signaling pathway utilized by human PMN in response to IL-2 may thus involve the association of lyn with IL-2Rbeta and the activation process also triggers the recruitment and activation of a specific ERK.

Functional role of phosphatidylinositol 3-kinase in direct tumor lysis by human natural killer cells.

Cytotoxicity is a key function of natural killer (NK) and T cells; yet the molecular mechanism is unclear. We have biological, biochemical and molecular evidence to demonstrate that phosphatidyl-inositol (PI) 3-kinase is critical for direct NK lysis of tumor cells, via control of intracellular granule movement. Tumor cell engagement rapidly activated PI 3-kinase in NK cells within 5 min, as demonstrated by p85 subunit tyrosine phosphorylation and its ability to generate phosphatidylinositol 3-phosphate, PI(3)P, from PI. Wortmannin and LY294002 effectively inhibited NK cells to lyse 51Cr-labeled tumor cells at the same doses that blocked PI-phosphorylating function in tumor-activated NK cells. Immunostaining demonstrated that tumor engagement for only 5 min mobilized perforin and granzyme B from NK cells unidirectionally towards the target, and prior treatment of NK cells with either PI 3-kinase inhibitor effectively stopped this intracellular polarization. Lastly, ectopic expression of dominant-negative p85 or p110 mutant markedly suppressed NK lytic capacity. These results taken together demonstrate that PI 3-kinase may control NK lytic function via granule polarization towards the contacted target cell.