The kinase anchoring protein Gravin has been designated as a tumor suppressor because the human gene resides in the q24-25.2 locus of chromosome 6, a deletion hotspot in advanced prostate, breast, and ovarian cancers (Xia et al., 2001
). In addition, overexpression of the murine ortholog SSeCKS protects against anoikis in v-Src
-transformed fibroblasts (Lin et al., 1996
). Although both observations insinuate that Gravin might repress tumor growth, a growing body of evidence calls into question whether Gravin constitutes a bona fide tumor suppressor. For example, SSecks null mice are viable, and while they exhibit benign prostatic hyperplasia, they do not develop tumors (Akakura et al., 2008
). Furthermore, gene array and proteomic analyses show that Gravin is upregulated in several transformed cell lines and cancers including chronic myelogenous leukemia, bladder cancer, and follicular lymphomas (Jiang et al., 1997
; Tsujimoto et al., 2005
; Zhu et al., 2002
). In this report we define an alternate role for Gravin in the spatial and temporal organization of phosphorylation-dependent protein-protein interactions that sustain mitosis. Several lines of evidence support this claim. While gene silencing of Gravin modestly increases tumor growth in immunodeficient mice, a more important consideration is that Gravin-depleted tumors have an elevated mitotic index. This may occur because Gravin depletion causes cells to stall during mitosis. This latter notion tallies with live-cell imaging and spindle profile studies that detect a prometaphase stall upon shRNA knockdown of Gravin in HeLa cells. Importantly, re-expression of Gravin rescued this defect. Thus, rather than solely exerting a dampening effect on mitosis, we propose that Gravin may augment the fidelity and reproducibility of cell division.
Protein phosphorylation by cyclin-dependent kinases (CDKs) drives eukaryotic cell division (Vermeulen et al., 2003
). Our mass spectrometry analysis establishes that Gravin is phosphorylated at numerous sites during mitosis. Deeper biochemical analyses reveal that CDK1 phosphorylation of Thr766 on Gravin earmarks a subpopulation of the scaffolding protein for a specialized role in cell division via the recruitment of Plk1. A defining characteristic of Plk1 is the ability to transiently associate with different mitotic structures as cells progress through mitosis (Petronczki et al., 2008
). Through mechanisms that are incompletely understood, these synchronized movements proceed through a C-terminal phosphopeptide-binding PBD that directs Plk1 toward its substrates and binding partners (Elia et al., 2003
). Our findings argue that phospho-T766 Gravin participates in the sequestering of Plk1. However, depletion of Gravin was not found to substantially affect the localization of Plk1 (Figures S5I–S5R
Immunofluorescence analysis in HEK293 and Rat-2 cells reveals that Gravin and Plk1 transiently associate during the early stages of mitosis. Both proteins are intermingled with DNA at prophase. This location is consistent with reports that Plk1 activity is necessary for chromosomal segregation (Jeong et al., 2010
). Live-cell imaging experiments show that gene silencing of Gravin correlates with a higher incidence of mitotic abnormalities. These include protracted prometaphase, misalignment of chromosomes on the metaphase plate, and an increased prevalence of aneuploidy. Interestingly, analogous patterns of prometaphase arrest and the initiation of spindle assembly checkpoints are observed in cells treated with the Plk1 selective inhibitor BI2536 (Brennan et al., 2007
; Petronczki et al., 2007
). Likewise, elegant chemical-genetic approaches that acutely inactivate the kinase at the metaphase-to-anaphase transition show that Plk1 activity is necessary to initiate cytokinesis, a process that completes cell division to yield two physically distinct daughter cells (Burkard et al., 2007
). Ancillary support for this latter scenario is highlighted by data showing that phospho-T766 Gravin staining remains at the centrosomes, whereas the Plk1 signal accumulates at the spindle midzone as cells enter anaphase. The segregation of these signals at later stages of mitosis not only emphasizes the precise temporal control that surrounds the Gravin-Plk1 interaction but also illustrates the remarkable mobility of this mitotic kinase. Thus, transient association with phospho-T766 Gravin offers a previously unrecognized means to direct Plk1 activity during the early phases of mitosis. Conversely, loss of this protein-protein interaction is deleterious to mitotic progression, as depletion of Gravin or overexpression of the nonphosphorylatable Gravin T766A mutant retards cell growth.
Grade 4 astrocytomas, more commonly known as glioblastoma, are extremely aggressive brain tumors (Van Meir et al., 2010
). The median survival time from diagnosis is approximately 12 months, and standard treatment (surgery, radiotherapy, chemotherapy, and antiangiogenic drugs) only extends life expectancy by 5–7 months (Krex et al., 2007
). Therefore the development of new prognostic markers for glioblastoma is critical. We show that phospho-T766 Gravin antiserum identifies rapidly dividing cells in paraffin-embedded sections from resected glioblastomas. Plk1 mRNA levels also increase in glioblastoma and correlate with both tumor classification and the probability of tumor recurrence after therapeutic intervention (Cheng et al., 2012
). Thus, detection of the Gravin-Plk1 complex may have prognostic value; however, evaluation of many more clinical samples will be necessary to firmly establish this postulate. In conclusion, our discovery of this dynamic kinase-anchoring event underscores the exquisite molecular constraints required for cell-cycle progression. Moreover, our histological evaluation of phospho-T766 Gravin and Plk1 in clinical samples and dividing tumor cells leads us to speculate that aberrant control of this transient scaffolding event may have pathological ramifications.