Investigation of mechanisms for the Cofilin activation in metastatic tumor cells

Abstract

Metastasis is the direct cause of death in cancer patients. Chemotaxis is an essential factor in metastasis The earliest event in chemotaxis is setting the direction of cell migration. This event is defined as directional sensing and is initiated by detecting a chemoattractant gradient. Actin severing protein Cofilin has emerged as playing an essential role in actin dynamics at the plasma membrane during cell protrusion in response to chemoattractant. It has been demonstrated that the actin-severing activity of cofilin can be down-regulated by LIM kinase (LIMK)-dependent phosphorylation at serine 3. Chemotactic stimulaton in various cell types induces cofilin dephosphorylation, suggesting that cofilin activation in these cells occurs by a dephosphorylation mechanism. However, resting metastatic carcinoma cells have the majority of their cofilin in a dephosphorylated but largely inactive state. EGF stimulation induces an increase in cofilin activity at 60 sec along with an increase in phospho-cofilin indicating that cofilin dephosphorylation is not coupled to cofilin activation in these cells. Suppression of LIMK function by inhibiting Rho-associated protein kinase (ROCK) or LIMK siRNA inhibited the EGF-induced cofilin phosphorylation but had no effect on cofilin activity or lamellipod protrusion induced by EGF. Correlation analysis revealed that cofilin, p-cofilin, and LIMK are not co-localized and changes in the location of these proteins upon EGF stimulation indicate that they are not functionally coupled. Phospholipase C, which has been implicated in cofilin activation following EGF stimulation, does not regulate p-cofilin levels following EGF. Therefore, our results do not support a model for the initial activation of cofilin by dephosphorylation in response to chemoattractant stimulation in metastatic carcinoma cells. PIP2 is hypothesized as the inhibiting factor for cofilin activity in metastatic tumor cells. Using antibody, membrane fractionation and FRET it is demonstrated that after EGF stimulation, there is large decrease of PIP2 levels at the membrane and this is PLC regulated. It is demonstrated that cofilin is released into the cytoplasm and is active upon PIP2 hydrolysis. Hence, it is hypothesized that active PLC stimulates localized cofilin activity in response to an EGF gradient to initiate the directional sensing.;Key words: LIMK, cofilin-servering, ROCK, PIP2, PLC.