Signal Transduction Mechanisms

Edward Y. Skolnik, MD

Signal Transduction Mechanisms

Phosphatidylinositol 3-kinase (Ptdins-3K) is an important enzyme in signal transduction pathways and regulates a variety of biological responses including glucose uptake, cell survival, cell proliferation, motility, endocytosis, vesicle trafficking, and autophagy. Ptdins-3K mediates these responses by generating the second messengers Ptdins(3)P, Ptdins(3,4)P2, and Ptdins(3,4,5)P3. which then activate downstream signaling responses by mediating protein-lipid interactions; Ptdins(3,4)P2, and Ptdins(3,4,5)P3 activate downsteam signaling molecules by binding a subset of proteins with pleckstrin homology (PH) domains and Ptdins(3)P functions by binding a subset of proteins with PX or FYVE domains.

Function and regulation of myotubularins (MTM), a family of Ptdins 3-phosphatases

Myotubularins constitute a large family of lipid phosphatases that specifically dephosphorylate Ptdins(3)P. MTM1 is mutated in X-linked myotubular myopathy and MTMR2, and MTMR13 are mutated in Charcot-Marie-Tooth disease (CMT type 4B), although the mechanisms whereby MTM dysfunction leads to these diseases is unknown. Moreover, little is known about the general mechanism(s) whereby MTMs are regulated, or the specific biological processes regulated by the different MTMs.  We have undertaken several different approaches to understand the mechanisms whereby loss of MTMs lead to disease as well the mechanisms whereby MTMs are regulated and function to specifically regulate distinct downstream targets of Ptdins(3)P. 
Our initial studies of MTMs in the nematode C. elegans provided the first genetic evidence that MTMs function non-redundantly to dephosphorylate Ptdins(3)P and negatively regulate the Class III Ptdins3K. In addition, we identified 2 MTMs (MTMR6 and MTMR9) that function together as a heterodimer and play an essential role in endocytosis in C. elegans.
We recently identified several proteins that bind specifically to a single class of MTMs and which are specifically regulated by that MTM. One MTM target we identified is a Ca2+-activated K+ channel, KCa3.1 (also known as IKCa2+ or SK4).  KCa3.1 plays important roles in controlling proliferation by T cells by helping to maintain a negative membrane potential thereby facilitating Ca2+ influx. We found that KCa3.1 channel activity requires Ptdins(3)P and that MTMR6 inhibits Ptdins(3)P by dephosphorylating the 3’ position of Ptdins(3)P, leading to decreased Ptdins(3)P levels. Moreover, by inhibiting KCa3.1, MTMR6 functions as a negative regulator of Ca2+ influx and proliferation of human CD4 T cells pointing to a new and unexpected role for Ptdins(3)P and the Ptdins(3)P phosphatase MTMR6 in the negative regulation of CD4 T cells. Work is ongoing to examine the in vivo role of MTMR6 in T cell function as well as the mechanisms whereby Ptdins(3)P regulates KCa3.1 channel activity.

Elucidate the function of novel PI3K targets

To understand additional mechanisms whereby PI3-kinase regulates these responses, we identified and cloned several new Ptdins-3K targets and are working to elucidate the function of these proteins in Ptdins-3K signaling. In this regard, we recently studied mice deficient in BAM32 (B Cell Adaptor Molecule of 32 kDa), one of the PH-domain containing molecules we identified. We found that Bam32 was essential for normal B-cell proliferation following B cell receptor (BCR) activation and in generating T-independent II responses, the latter leading to a markedly increased susceptibility to infection by Streptococcus pneumonia We are now working to identify the signaling pathways regulated by BAM32 and whether mutation in BAM32 may account for increased susceptibility to encapusulated organisms in a subset of immunodeficient patients.

Function and regulation of Sterile (Ste 20) serine/threonine kinases

We previously identified a Ste20 kinase that interacts with the SH3 domains of Nck, termed NIK (Nck Interacting Kinase). We found that NIK and its Drosophila ortholog termed misshapen (msn) specifically activate the JNK map kinase pathway and are essential genes in development. Using genetics in Drosophila and experiments in mammalian cells and knock out mice, we have mapped out some of the pathways in which NIK and msn function. We are currently continuing to use these systems to further understand the biological roles of this family of kinases. More recently, we have begun to study the role of NIK in cancer. NIK protein expression is upregulated in a number of cancers and may contribute to oncogenesis by integrating growth signals from integrins.