Broad and structurally conserved patterns of tyrosine phosphorylation in secreted proteins in vivo have been widely identified in the existing literature but the kinase(s) responsible for these phosphorylations have been obscure, precluding functional analyses of these modifications. Our laboratory has recently identified the first known secreted protein tyrosine kinase (VLK). VLK is first expressed in the early epiblast, and is essential for normal organogenesis. We find that VLK phosphorylates a wide range of secreted proteins with established roles in the regulation of secretion, extracellular matrix (ECM) formation, and ECM remodeling. In many cases, sites phosphorylated by VLK correspond to sites of tyrosine phosphorylation in secreted proteins previously found to occur in vivo. We have also found that, during stimulated platelet secretion, VLK is co-released with endogenous ATP to drive de novo tyrosine phosphorylation outside the cell. These finding open up a broad new area in the study of dynamic regulation of ECM and other secreted proteins. In this proposal, we plan to investigate the regulation of VLK within the secretory pathway, identify secreted targets for VLK phosphorylation in cells that express VLK endogenously, and begin to establish how VLK phosphorylation modifies the function of specific secreted or secretory pathway resident proteins. We will examine in detail the components necessary for regulation of VLK secretion and phosphorylation, both in platelets in response to physiological stimuli, and in immortalized cells that secrete VLK either constitutively or in response to stimuli. We will examine the phosphorylated secretome in each of these cell types, and the dependence of these phosphorylations on VLK. We will then build on this identification of physiological VLK substrates in differentiated cell types to establish how VLK modifies specific substrate functions. These studies will define a new mechanism for the dynamic regulation of the activity of proteins in the secretory pathway and in the extracellular environment.
Diseases associated with pathological changes of the extracellular matrix have a tremendous impact on the health and wellbeing of all US citizens. These changes can include tissue remodeling during tumor metastasis, organ fibrosis, and atherosclerosis. We have identified a new mechanism for the regulation of extracellular matrix homeostasis and remodeling, with substantial implications for new therapeutic approaches to human disease pathology.
