Title

Altered signal transduction during mitosis

Date of Completion

January 2000

Keywords

Biology, Cell

Degree

Ph.D.

Abstract

Endocytosis, exocytosis, intracellular calcium transients, and arachidonic acid mobilization are all suppressed during mitosis. These phenomena have given rise to the hypothesis that membrane functions of mitotic cells are impaired, and that as a consequence, these cells may be insulated from external stimuli. I have examined this directly. I first focused on the activation of the EGF receptor, a plasma membrane receptor tyrosine kinase that specifically binds EGF and TGF-α. In the mitotic cell, the EGF receptor was autophosphorylated in response to either ligand, although phosphorylation was reduced compared to interphase levels. The mitotic receptor underwent subsequent dephosphorylation if bound to TGF-α, but, in contrast to interphase cells, when the receptor was bound to EGF, dephosphorylation did not occur. Furthermore, in mitotics, the adapter protein GRB2 moved to the membrane fraction when stimulated by TGF-α but remained in the cytosolic fraction in EGF-stimulated cells. ^ Because of the known susceptibility of mitotic cells to stress, I also asked whether activation of the stress pathway, which can be protective in interphase cells, might be inhibited in mitotics. Alternatively, it was possible that the pathway was already activated, as though mitosis itself were a physiological “stress”. I determined that p38 MAP kinase, a key enzyme in the stress pathway, was not constitutively active, nor could it be activated by a wide variety of stimuli. This suggested that the sensitivity of mitotic cells to stress might be a consequence of p38 inhibition. However, the present study, like most biochemical studies on mitotic cells, used an anti-microtubule agent (in our case colcemid) to arrest cells in metaphase. I found that long-term exposure to this drug profoundly inhibited p38 activation. Conditions were therefore developed such that colcemid itself had little effect on the kinase, and importantly, p38 activation was still inhibited during mitosis. These studies suggest that signal transduction is altered during mitosis in such a way that selective suppression of growth factor or stress pathway components could modify the function of mitotic cells, and potentially affect the character of the resulting daughter cells. ^