The focal adhesion scaffolding protein HEF1 regulates activation of the Aurora-A and Nek2 kinases at the centrosome

Abstract

Although HEF1 has a well-defined role in integrin-dependent attachment signaling at focal adhesions, it relocalizes to the spindle asters at mitosis. We report here that overexpression of HEF1 causes increase in centrosome numbers and multipolar spindles, resembling defects induced by manipulation of the mitotic regulatory kinase Aurora A (AurA). We show that HEF1 associates with and controls activation of AurA. We also show HEF1 depletion causes centrosomal splitting, monoastral spindles, and hyperactivation of Nek2, implying additional action earlier in cell cycle. These results provide new insights into the role of an adhesion protein in coordination of cell attachment and division. KeywordsHEF1; centrosome; spindle; mitosis; Aurora-A; Nek2 HEF1 is a member of a group of scaffolding proteins that includes p130Cas and Efs/Sin 1,2 . This group of Cas proteins localizes to focal adhesions in interphase cells, and acts as intermediates in a variety of integrin-dependent signaling processes, including establishment of cell attachments, migration, and cell survival signaling. In 1998, we proposed that HEF1 might have a previously unsuspected function in mitosis 3 , based on the observation that the HEF1 protein relocalized from focal adhesions to the mitotic spindle asters in M-phase. Since that time, reports have appeared suggesting of the association of other focal adhesion complex proteins, such as zyxin 4 , paxillin 5 , FAK, and Pyk2 6 with the mitotic spindle or other relevant structures such as the microtubule organizing center (MTOC) or centrosome. Recent work has emphasized the dual activity of centrosomes in contributing to control of cell polarization in interphase cell migration 7,8 , but also in coordinating assembly of the mitotic spindle in Mphase 9 . Centrosomally-associated signaling activities such as the Aurora-A (AurA) kinase also govern the timing of mitotic entry 10 , for instance by regulating the activation of cyclin B1 11 . In this study, we demonstrate an requirement for HEF1 in activation of AurA and Nek2 12 , a second kinase important for centrosome cohesion, and we provide additional data indicating HEF1 provides a crucial bridge coordinating cell attachment and cell division processes in mammalian cells. Results Cell cycle-regulated HEF1 localizationHEF1 localizes to the centrosome of MCF7 cells in a cell cycle regulated manner ( Figure 1A), with centrosomal signal lowest in G1, and strongest in G2/M cells. This corresponds to * corresponding author: Erica Golemis, W406, Fox Chase Cancer Center, 333 Cottman Ave., Philadelphia, PA 19111, Phone: 215-728-2860, Fax: 215-728-3616 Figure 2A; 3). As HEF1 levels increase during G2, a slower migrating, hyperphosphorylated species becomes more apparent, as we have previously reported 3 . At mitotic entry, a significant fraction of HEF1 moves to the spindle, and HEF1 is no longer detectable at the centrosome at cytokinesis. The endogenous HEF1 localization pattern described here was lost following HEF1 depletion with siRNA, supporting si...

Phosphorylation of human enhancer of filamentation (HEF1) on serine 369 induces its proteasomal degradation

“…It has been reported that HEF1 phosphorylation is cell cycle-regulated and that HEF1 is a substrate for Aurora-A kinase [22]. The above experiment showed that OA treatment revealed at least two different states of phosphorylation of HEF1.…”

“…So far, only the Aurora-A kinase was identified as an HEF1 kinase in MCF-7 cells, targeting ser-296. HEF1 interaction with Aurora-A during mitosis leads to kinase activation and its dissociation from phosphorylated HEF1 [22].…”

“…Serine 296 of HEF1 has been described as a target amino acid for Aurora-A phosphorylation [22]. In addition, the examination of the primary sequence of the protein revealed the existence of an R-X-S located at serine 369 matching the consensus phosphorylation site for Aurora-B but also for other kinases which may be inhibited by Hesperadin [31].…”

“…However, the serine/threonine kinase(s) responsible for this HEF1 phosphorylation have not been identified in most cases. A recent study has described the phosphorylation of serine 296 of HEF1 by the cell-cycle-dependent kinase Aurora-A [22]. Several reports pointed out a tyrosine dephosphorylation of the components of the focal adhesion complex including FAK, paxillin and p130 Cas associated with ser/thr phosphorylation of these proteins and the dissociation of the complex during the M phase of the cell cycle.…”

“…Aurora-A activity is down-regulated by dephosphorylation of the threonine 288 by the ser/thr phosphatase PP1 [29]. HEF1 interaction with Aurora-A leads to activation of the kinase while phosphorylated HEF1 fate is still unclear [22]. Aurora-B binds to and is negatively regulated by both PP1 and PP2A and is therefore activated by okadaic acid (OA), an inhibitor of these phosphatases [30].…”