Erik W. Wilker

Merlin, the Product of the Nf2 Tumor Suppressor Gene, Is an Inhibitor of the p21-Activated Kinase, Pak1

The Nf2 tumor suppressor gene codes for merlin, a protein whose function has been elusive. We describe a novel interaction between merlin and p21-activated kinase 1 (Pak1), which is dynamic and facilitated upon increased cellular confluence. Merlin inhibits the activation of Pak1, as the loss of merlin expression results in the inappropriate activation of Pak1 under conditions associated with low basal activity. Conversely, the overexpression of merlin in cells that display a high basal activity of Pak1 resulted in the inhibition of Pak1 activation. This inhibitory function of merlin is mediated through its binding to the Pak1 PBD and by inhibiting Pak1 recruitment to focal adhesions. This link provides a possible mechanism for the effect of loss of merlin expression in tumorigenesis.

Structural and Functional Versatility of the Fha Domain in DNA-Damage Signaling by the Tumor Suppressor Kinase Chk2

The Chk2 Ser/Thr kinase plays crucial, evolutionarily conserved roles in cellular responses to DNA damage. Identification of two pro-oncogenic mutations within the Chk2 FHA domain has highlighted its importance for Chk2 function in checkpoint activation. The X-ray structure of the Chk2 FHA domain in complex with an in vitro selected phosphopeptide motif reveals the determinants of binding specificity and shows that both mutations are remote from the peptide binding site. We show that the Chk2 FHA domain mediates ATM-dependent Chk2 phosphorylation and targeting of Chk2 to in vivo binding partners such as BRCA1 through either or both of two structurally distinct mechanisms. Although phospho-dependent binding is important for Chk2 activity, previously uncharacterized phospho-independent FHA domain interactions appear to be the primary target of oncogenic lesions.

A structural basis for 14-3-3sigma functional specificity.

The 14-3-3 family of proteins includes seven isotypes in mammalian cells that play numerous diverse roles in intracellular signaling. Most 14-3-3 proteins form homodimers and mixed heterodimers between different isotypes, with overlapping roles in ligand binding. In contrast, one mammalian isoform, 14-3-3σ, expressed primarily in epithelial cells, appears to play a unique role in the cellular response to DNA damage and in human oncogenesis. The biological and structural basis for these 14-3-3σ-specific functions is unknown. We demonstrate that endogenous 14-3-3σ preferentially forms homodimers in cells. We have solved the x-ray crystal structure of 14-3-3σ bound to an optimal phosphopeptide ligand at 2.4 Å resolution. The structure reveals the presence of stabilizing ring-ring and salt bridge interactions unique to the 14-3-3σ homodimer structure and potentially destabilizing electrostatic interactions between subunits in 14-3-3σ-containing heterodimers, rationalizing preferential homodimerization of 14-3-3σ in vivo. The interaction of the phosphopeptide with 14-3-3 reveals a conserved mechanism for phospho-dependent ligand binding, implying that the phosphopeptide binding cleft is not the critical determinant of the unique biological properties of 14-3-3σ. Instead, the structure suggests a second ligand binding site involved in 14-3-3σ-specific ligand discrimination. We have confirmed this by site-directed mutagenesis of three σ-specific residues that uniquely define this site. Mutation of these residues to the alternative sequence that is absolutely conserved in all other 14-3-3 isotypes confers upon 14-3-3σ the ability to bind to Cdc25C, a ligand that is known to bind to other 14-3-3 proteins but not to σ.

14-3-3σ controls mitotic translation to facilitate cytokinesis

14-3-3 proteins are crucial in a wide variety of cellular responses including cell cycle progression, DNA damage checkpoints and apoptosis. One particular 14-3-3 isoform, σ, is a p53-responsive gene, the function of which is frequently lost in human tumours, including breast and prostate cancers as a result of either hypermethylation of the 14-3-3σ promoter or induction of an oestrogen-responsive ubiquitin ligase that specifically targets 14-3-3σ for proteasomal degradation. Loss of 14-3-3σ protein occurs not only within the tumours themselves but also in the surrounding pre-dysplastic tissue (so-called field cancerization), indicating that 14-3-3σ might have an important tumour suppressor function that becomes lost early in the process of tumour evolution. The molecular basis for the tumour suppressor function of 14-3-3σ is unknown. Here we report a previously unknown function for 14-3-3σ as a regulator of mitotic translation through its direct mitosis-specific binding to a variety of translation/initiation factors, including eukaryotic initiation factor 4B in a stoichiometric manner. Cells lacking 14-3-3σ, in marked contrast to normal cells, cannot suppress cap-dependent translation and do not stimulate cap-independent translation during and immediately after mitosis. This defective switch in the mechanism of translation results in reduced mitotic-specific expression of the endogenous internal ribosomal entry site (IRES)-dependent form of the cyclin-dependent kinase Cdk11 (p58 PITSLRE), leading to impaired cytokinesis, loss of Polo-like kinase-1 at the midbody, and the accumulation of binucleate cells. The aberrant mitotic phenotype of 14-3-3σ-depleted cells can be rescued by forced expression of p58 PITSLRE or by extinguishing cap-dependent translation and increasing cap-independent translation during mitosis by using rapamycin. Our findings show how aberrant mitotic translation in the absence of 14-3-3σ impairs mitotic exit to generate binucleate cells and provides a potential explanation of how 14-3-3σ-deficient cells may progress on the path to aneuploidy and tumorigenesis.

ROS Fusion Tyrosine Kinase Activates a SH2 Domain–Containing Phosphatase-2/Phosphatidylinositol 3-Kinase/Mammalian Target of Rapamycin Signaling Axis to Form Glioblastoma in Mice

Glioblastoma multiforme is the most common and lethal form of primary brain cancer. Diagnosis of this advanced glioma has a poor prognosis due to the ineffectiveness of current therapies. Aberrant expression of receptor tyrosine kinases (RTK) in glioblastoma multiformes is suggestive of their role in initiation and maintenance of these tumors of the central nervous system. In fact, ectopic expression of the orphan RTK ROS is a frequent event in human brain cancers, yet the pathologic significance of this expression remains undetermined. Here, we show that a glioblastoma-associated, ligand-independent rearrangement product of ROS (FIG-ROS) cooperates with loss of the tumor suppressor gene locus Ink4a;Arf to produce glioblastomas in the mouse. We show that this FIG-ROS-mediated tumor formation in vivo parallels the activation of the tyrosine phosphatase SH2 domain-containing phosphatase-2 (SHP-2) and a phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling axis in tumors and tumor-derived cell lines. We have established a fully penetrant preclinical model for adult onset of glioblastoma multiforme in keeping with major genetic events observed in the human disease. These findings provide novel and important insights into the role of ROS and SHP-2 function in solid tumor biology and set the stage for preclinical testing of targeted therapeutic approaches.

Validation of the p21-Activated Kinases as Targets for Inhibition in Neurofibromatosis Type 2

Neurofibromatosis type 2 (NF2) is a dominantly inherited cancer disorder caused by mutations at the NF2 gene locus. Merlin, the protein product of the NF2 gene, has been shown to negatively regulate Rac1 signaling by inhibiting its downstream effector kinases, the p21-activated kinases (Pak). Given the implication of Paks in tumorigenesis, it is plausible that merlins tumor suppressive function might be mediated, at least in part, via inhibition of the Paks. We present data indicating this is indeed the case. First, analysis of primary schwannoma samples derived from NF2 patients showed that in a significant fraction of the tumors, the activity of Pak1 was highly elevated. Second, we used shRNAs to knockdown Pak1, 2, and 3 in NIH3T3 cells expressing a dominant-negative form of merlin, NF2(BBA) (NIH3T3/NF2(BBA)), and find that simultaneous knockdown of Pak1-3 in these cells significantly reduced their growth rates in vitro and inhibited their ability to form tumors in vivo. Finally, while attempting to silence Pak1 in rat schwannoma cells, we found that these cells were unable to tolerate long-term Pak1 inhibition and rapidly moved to restore Pak1 levels by shutting down Pak1 shRNA expression through a methylation-dependent mechanism. These data suggest that inhibiting Pak could be a beneficial approach for the development of therapeutics toward NF2. In addition, the finding that the shRNA-mediated Pak1 suppression was silenced rapidly by methylation raises questions about the future application of such technologies for the treatment of diseases such as cancer.