Identification and Characterization of a Novel Posttranslational Modification of Merlin in Tumor Suppression

Background: Neurofibromatosis type 2 (NF2) is a genetic disease in which patients may develop multiple tumors in their nervous system, such as schwannoma and meningioma. Despite major advances in neuroimaging and neurosurgical techniques over the past decades, neurosurgical management of NF2 patients remains challenging. In order to alter the clinical outcome of NF2, it is important to develop targeted therapies based on the identified genetic defects driving its development and sustaining its maintenance.

It is known that this disease is caused by defects in a gene called NF2, which produces a protein called Merlin. In tumors associated with this disease, Merlin is frequently found to be dysfunctional. Studies have established that Merlin is a multi-functional protein. It can activate the Hippo tumor suppressor pathway or inhibit various tumorigenic signals. However, how Merlin achieves these multiple tasks in a cell is still not clear. Answering this question is important to developing effective targeted therapies. One way to resolve this issue is to use a model in which Merlin activation and function can be delineated in a timely manner or, ideally, in real time.

Objective/Hypothesis: Recently, we uncovered a link between calcium and Merlin activation. We found that increasing calcium concentration in cells by various methods activates Merlin through a novel protein modification. This modification happens through attachment of a small protein, called ubiquitin, to Merlin. This so-called mono-ubiquitylation process usually modifies protein activities. We found that such mono-ubiquitylation occurs within a few minutes after calcium concentration was increased in cells. Importantly, we found that such Merlin mono-ubiquitylation is not only associated with calcium increase, but also exists in other physiological conditions when Merlin is active. Therefore, this system provides us a simple but very sensitive model to delineate Merlin activation by mono-ubiquitylation and examine the function of this modification. Based on our preliminary results, the objective of this proposed study is to further functionally and mechanistically examine the novel protein modification of Merlin using the unique calcium-induced model and other physiological models. We hypothesize that this modification of Merlin is essential for its function in tumor suppression, and in some NF2-related tumors, such modification has been lost.

Impact: Merlin is a multi-functional protein. How Merlin achieves these multiple tasks in a cell is still not clear. This is an obstacle in the field to understanding the nature of Merlin dysfunction in NF2. Here, we uncovered that calcium is a novel cellular activator of Merlin. Using this unique calcium-induced system, we further uncovered mono-ubiquitylation as a novel post-translational modification during Merlin activation. By further studying this modification, we will demonstrate a mechanism by which Merlin is activated and delineate the steps of Merlin activation and function. Our studies will also shed light on how Merlin is dysfunctional in NF2 and provide avenues for development of more effective therapies. Therefore, this proposed research will specifically address one of the FY17 NFRP Areas of Emphasis: Target identification, drug discovery, and targeted and immunotherapies.