[Frontiers in Bioscience 13, 528-539, January 1, 2008] 528 MT-SP1 proteolysis and regulation of cell-microenvironment interactions Molly R Darragh 1 , Ami S Bhatt 2 , Charles S Craik 3 1 Graduate Program in Biophysics, University of California, San Francisco, 2 Brigham and Women's Hospital, Department of Internal Medicine, 75 Francis Street, Boston, MA 02115, 3 Department of Pharmaceutical Chemistry, University of California, San Francisco TABLE OF CONTENTS 1. Abstract 2. Introduction 3. MT-SP1 and substrates 3.1. Identifying putative substrates 3.2. Protease activation 3.3. Growth factor activation 3.4. Receptor activation 3.5. Additional substrates 3.6. VEGFR-2 is inactivated by MT-SP1 4. Discussion 4.1. MT-SP1 and cancer 4.2. Substrate validation 5. Experimental Methods 5.1. Recombinant VEGFR-2 cleavage reactions 5.2. VEGFR-2 cleavage and ERK1/2 activation assay 6. Acknowledgements 7. References 1. ABSTRACT MT-SP1 is a type II transmembrane serine protease implicated in a range of human cancers including those of the breast, cervix, ovaries, prostate, colon and gastrointestinal tract. Mouse models have shown it to be critical for proper epidermal development and postnatal survival. However, the role of this enzyme in normal and malignant biology has not yet been fully elucidated. Several groups have identified putative substrates of MT- SP1 in an effort to understand the possible biological processes in which this protease may be involved. Methods for substrate identification include comparing known protein cleavage sequences with MT-SP1 specificity data, in vitro cleavage assays, examining genetic microarrays for enzyme/substrate coexpression, immunohistochemistry for colocalization, and a variety of phenotypic observations using cell culture and mouse models. Given the inherent limitations of each individual method, substrate plausibility is best substantiated using a combination of experimental approaches. Here we review MT-SP1 substrates identified to date and the possible physiological implications of substrate cleavage in cell-microenvironment interactions. This data indicates that MT-SP1 is capable of playing roles in growth factor activation, receptor activation and inactivation, protease activation, and ectodomain shedding. We also present for the first time vascular endothelial growth factor receptor 2 (VEGFR-2) as a putative substrate for MT-SP1. 2. INTRODUCTION Proteolysis is a critical step in many biological processes from protein degradation to viral maturation and cell cycle progression(1-3). Proteases often mediate these processes by cleaving proteins involved in signaling pathways. The high processivity of proteases makes them well suited to amplify signals within a pathway, as in angiogenesis or the blood coagulation cascade (4-6). Unlike some enzyme pairs, such as the kinase/phosphatases, proteases are not paired with protein ligases. Proteolysis is thus often a “one way” signaling event that must be regulated by protein expression, localization, activation and inhibition. This rapid, tunable, and irreversible method of protein modification allows cells to regulate a number of degradative processes and signaling pathways. Spatial distribution and confinement of proteases ensures that the correct signals are processed in the correct environment. Proteolysis which is localized to the cell surface is of particular interest in that it can be crucial in orchestrating cell-environment interactions (7). This may include cellular responses to the environment, as when growth factors and/or receptors are modified extracellularly to initiate intracellular signaling, or it may be used to modify the environment in response to cellular signals, such as extracellular matrix (ECM) degradation.