Cell cycle-regulated processing of HEF1 to multiple protein forms differentially targeted to multiple subcellular...

Current research has established that HEF1 and related proteins coordinate cell adhesion through multidomain structures. These proteins interact with focal adhesion kinase via SH3 domains and are phosphorylated by oncoproteins like Abl and Fyn. However, the mechanisms governing HEF1 processing and its subcellular targeting remain unclear. Prior studies have focused on lymphoid cells, leaving a gap in understanding HEF1's role in epithelial tissues. The regulation of HEF1 expression during the cell cycle has not been fully characterized. No prior work has resolved the functional implications of HEF1 cleavage products. This uncertainty drives the need to investigate HEF1 processing and localization in diverse cell types. The connection between HEF1 and spindle regulation is an emerging area of inquiry. Understanding these aspects could clarify HEF1's role in cancer progression.

This study aims to explore how HEF1 is processed into multiple protein forms and how these forms are targeted to different subcellular compartments. The specific problem involves understanding the cell cycle regulation of HEF1 and its localization in epithelial cells. The motivation stems from the lack of clarity on HEF1's functional diversity and its potential role in cancer. By analyzing HEF1 in breast and lung epithelial cells, the study expands beyond prior lymphoid cell research. The investigation includes examining HEF1's phosphorylation states and cleavage products. The goal is to determine how these forms are distributed in the cell and how they relate to cell cycle progression. The study also seeks to identify interacting proteins that could clarify HEF1's function. This approach addresses a gap in the literature on HEF1's role in cell cycle regulation and cancer.

The study uses transfection of a single HEF1 cDNA to generate multiple protein forms. Western blotting and immunoprecipitation are employed to identify HEF1 species. Phosphorylation states are analyzed using phospho-specific antibodies. Cleavage sites are identified through sequencing and caspase activity assays. Localization of HEF1 proteins is assessed using immunofluorescence in MCF-7 cells. Expression patterns are tracked across the cell cycle using synchronized cell cultures. Two-hybrid screening is used to find interacting proteins. The interaction between HEF1 and the G2/M spindle-regulatory protein Dim1p is confirmed through co-immunoprecipitation.

Transfection of HEF1 cDNA produces four protein forms: p115, p105, p65, and p55. p115 and p105 represent different phosphorylation states of full-length HEF1. p55 is derived from cleavage at a caspase consensus site in the HEF1 sequence. HEF1 is highly expressed in breast and lung epithelial cells, as well as lymphoid cells. In MCF-7 cells, p105 and p115 are upregulated during cell growth. p55 is produced specifically at mitosis. p105 and p115 localize to focal adhesions in the cytoplasm. p55 associates with the mitotic spindle, suggesting a role in spindle regulation.

The findings suggest that HEF1 is processed into multiple forms with distinct subcellular localizations. p105 and p115 are cytoplasmic and phosphorylation-dependent, while p55 is cleaved and mitosis-specific. HEF1's association with focal adhesions supports its role in cell adhesion. The mitotic localization of p55 implies a function in spindle regulation. The interaction with Dim1p supports this role. These data suggest that HEF1 may connect morphological signals with cell cycle regulation. The study highlights HEF1's potential involvement in cancer progression pathways. The results provide a foundation for further research into HEF1's functional diversity and regulatory mechanisms.