No abstract

Members of the membrane-type matrix metalloproteinases (MT-MMPs) have been implicated in a wide range of physiological and pathological processes from normal development to tumor growth. Tethered on plasma membrane, these enzymes are potentially regulated by the trafficking machinery of the cells. Here we demonstrate that both MT1-MMP and MT3-MMP are internalized, transported to the trans-Golgi network through early endosomes, and recycled back to cell surface in 60 min in a manner distinct from the one employed by transferrin receptor. Interestingly, co-expressed MT1-MMP and MT3-MMP are localized and routed in the same vesicles throughout the trafficking process. We further demonstrated that the carboxyl-terminal sequence DKV 582 of MT1-MMP is required for its recycling, thus defining a novel recycling motif. These results suggest that MTMMPs may coordinate their proteolytic activities through the cellular trafficking machinery.The cell surface, populated with various protein molecules that mediate communications between the cell and its immediate environment, in many ways defines the cellular phenotype. One class of such molecules is cell surface proteinases that exert functional influence through irreversible cleavage of their substrates (1-4). Therefore, these surface proteinases should be regulated properly, and perhaps, differently from other cell surface molecules. The membrane-type matrix metalloproteinases or MT-MMPs 1 are good examples of cell surface proteinases employed by various cell types to alter their surrounding environment during angiogenesis, tissue remodeling, tumor invasion, and metastasis (1,(5)(6)(7)(8)(9)(10). Displayed on the cell surface, these molecules have been shown to mediate a diverse biochemical reactions such as the activation of soluble MMPs, i.e. MMP-2 and MMP-13, or direct degradation of extracellular matrix components (6,(11)(12)(13)(14). Ablation of MT1-MMP in mice validated most of these functions in vivo (15,16). Yet little is known about the cellular mechanisms regulating the trafficking of MT-MMPs to and from the cell surface.The MT-MMP subfamily can be divided into two subgroups, MT1-, MT2-, MT3-, and MT5-MMPs with type I transmembrane domains and MT4-and MT6-MMPs with glycosylphosphatidylinositol anchors (6 -9, 18, 20). Because the first group MT-MMPs have similar structures at their carboxyl termini with transmembrane domains and cytoplasmic tails, it is expected that their activities should be regulated by the trafficking machinery that presumably interacts with their cytoplasmic tails (23,24). Given their apparent divergence in their cytoplasmic domains, the trafficking pattern of each MT-MMP may differ significantly (23). Indeed, although MT1-MMP and MT3-MMP are regulated by endocytosis, MT5-MMP does not appear to internalize well in the cells analyzed (24). Functionally, we have recently shown that MT1-MMP and MT3-MMP have distinct activities toward two well established substrates, proMMP-2 and type I collagen (25). Furthermore, these distinctions are encoded in...

Using a cell-based high-throughput screen designed to detect small chemical compounds that inhibit cell growth and survival, we identified three structurally related compounds, 21A8, 21H7, and 65D4, with differential activity on cancer versus normal cells. Introduction of structural modifications yielded compound M-110, which inhibits the proliferation of prostate cancer cell lines with IC 50 s of 0.6 to 0.9 μmol/L, with no activity on normal human peripheral blood mononuclear cells up to 40 μmol/L. Screening of 261 recombinant kinases and subsequent analysis revealed that M-110 is a selective inhibitor of the PIM kinase family, with preference for PIM-3. The prostate cancer cell line DU-145 and the pancreatic cancer cell line MiaPaCa2 constitutively express activated STAT3 (pSTAT3 , we used PIM-1-, PIM-2-, or PIM-3-specific siRNA and showed that knockdown of PIM-3, but not of PIM-1 or PIM-2, in DU-145 cells results in a significant downregulation of pSTAT3 Tyr705. The phosphorylation of STAT5 on Tyr694 in 22Rv1 cells is not affected by M-110 or SGI-1776, suggesting specificity for pSTAT3 Tyr705 . These results identify a novel role for PIM-3 kinase as a positive regulator of STAT3 signaling and suggest that PIM-3 inhibitors cause growth inhibition of cancer cells by downregulating the expression of pSTAT3 Tyr705