Integrin Activity in Disease
Jeroen Pouwels, PhD, Adjunct Professor in Biochemistry
Academy Of Finland Research Fellow
Email: jeroen.pouwels [at] utu.fi.
Research Coordinator of the TEHO project at the University of Helsinki (lab of Juha Klefström)
Email: jeroen.pouwels [at] helsinki.fi.
Turku Centre for Biotechnology
-Meraj Hasan Khan, MSc.
-Siiri Salomaa, MSc.
-Umar Butt, MSc.
Sharpin is a multifunctional adaptor protein that is amplified and overexpressed in a wide variety of human cancer types. Importantly, Sharpin promotes cancer cell proliferation and tumor formation and drives metastasis. In addition, high Sharpin levels correlate with poor clinical outcome in breast cancer, suggesting that Sharpin is a prognostic biomarker. However, how Sharpin promotes carcinogenesis and metastasis remains largely unknown.
Integrins are heterodimeric receptors that consist of a α- and β -subunit and are essential for cell adhesion to the extracellular matrix, which activates many intracellular signalling pathways. Regulation of integrin activity is fundamentally important during development and in many physiological processes, and deregulated integrin activation can cause many diseases, such as bleeding disorders, immune-deficiencies, chronic inflammation and cancer. Importantly, we identified Sharpin as an inhibitor of integrin activity (Rantala, Pouwels, et al., Nature Cell Biol. 2011), which opened a new paradigm in integrin regulation by showing that dynamic switching between the inactive and active conformation is controlled by a protein that interacts with the α-subunit cytoplasmic domain. More recently, we showed (Pouwels et al., Cell Rep. 2013) that Sharpin inhibits the lymphocyte-specific α Lβ2-integrin, which controls lymphocyte migration and allows lymphocyte detachment during transmigration.
Therefore, Sharpin-mediated integrin inhibition could explain some of the oncogenic and pro-metastasis properties of Sharpin. However, in addition to integrins, Sharpin binds several other signalling proteins and regulates their activity, suggesting that Sharpin acts as a multifunctional adaptor protein. For example, Sharpin is a component of the linear ubiquitination assembly complex (LUBAC) that promotes signal-induced activation of the oncogenic and pro-inflammatory transcription factor NF-κB, which mediates the cellular response to various stimuli. Furthermore, direct interaction of Sharpin with PTEN, one of the most commonly lost tumor suppressors in human cancer, inhibits PTEN activity.
Despite the recent discoveries in the linear ubiquitin field and the range of functional Sharpin interactors, a systematic approach to map individual proteins and signalling pathways regulated by Sharpin and/or LUBAC has not been reported. We have now recently determined the first ‘Sharpin interactome’, which identifies a large number of potential Sharpin interactors and suggests that Sharpin regulates a wide range of key biological processes, such as metabolism, RNA processing, vesicular transport and the actin cytoskeleton (Khan et al., under review). In addition, we confirm the novel direct interaction between Sharpin and the Arp2/3 complex, which forms the branched actin filaments that mediate a range of important cellular functions like cell migration and endocytosis. Using super-resolution microscopy we show that Sharpin and the Arp2/3 complex colocalize in lamellipodia, the leading edge of migrating cells. Furthermore, we identify residues in Sharpin that mediate Arp2/3 binding. Using a specific Arp2/3 binding-deficient Sharpin mutant we identify a novel role for Sharpin in lamellipodium formation and cell migration that depends on Sharpin interaction with the Arp2/3 complex but is independent of LUBAC function (Khan et al., Journal of Cell science, 2017).
Furthermore, using mass spectroscopy analyses of GFP-Sharpin, pulled down from cancer cells, and recombinant GST-Sharpin, subjected to in vitro kinase assays, we identified a number of conserved residues that are phosphorylated by oncogenic kinases. Mutation of a selection of these residues identified phosphorylation sites that critically regulate Sharpin-mediated Arp2/3 activation and LUBAC function without affecting Sharpin-mediated integrin inhibition. We now aim to develop phosphorylation-specific antibodies to further study what role these phosphorylation events play in Sharpin function and to identify the kinases that mediate these phosphorylation events. Furthermore, we will determine if Sharpin gets differentially phosphorylated under different experimental conditions.
In addition, as integrins and the Arp2/3 complex both play well-documented roles in cell invasion and metastasis, we are investigating whether Sharpin also plays a role in these processes.
Academy of Finland
(† Shared first-authorship)
Khan M, Salomaa S, Jacquemet G, Butt U, Miihkinen M, Deguchi T, Kremneva E, Lappalainen P, Humphries M, Pouwels J. The Sharpin interactome reveals a role for Sharpin in lamellipodium formation through the Arp2/3 complex. J. Cell Sc. 2017; 130, 3094-3107 doi:10.1242/jcs.200329.
Lilja J, Zacharchenko T, Georgiadou M, Jacquemet G, De Franceschi N, Peuhu E, Pouwels J, Beifuss M, Boeckers T, Kreienkamp H-J, Barsukov I, Ivaska J. SHANK3 structure reveals a Ras-associated domain regulating integrin activation. Nat Cell Biol. 2017:19(4):292-305.
De Franceschi N, Peuhu E, Parsons M, Rissanen S, Vattulainen I, Salmi M, Ivaska J*, Pouwels J*. Mutually Exclusive Roles of SHARPIN in Integrin Inactivation and NF-?B Signaling. PloS one. 2015;10(11): e0143423.
De Franceschi N, Arjonen A, Elkhatib N, Dennessiouk K, Wrobel A, Wilson T, Pouwels J, Montagnac G, Owen D, Ivaska J. Selective integrin endocytosis is driven by ? chain:AP2 interactions. Nat Struct Mol Biol. 2016; 23: 172–179.
Wang Y, Arjonen A, Pouwels J, Ta H, Pausch P, Bange G, Engel U, Pan X, Fackler OT, Ivaska J, Grosse R. Formin-like 2 Promotes beta1-Integrin Trafficking and Invasive Motility Downstream of PKCalpha. Dev Cell. 2015;34: 475-483.
Pouwels J†, De Franceschi N†, Rantakari P, Auvinen K, Karikoski M, Mattila E, Potter C, Sundberg JP, Hogg N, Gahmberg CG, Salmi M, Ivaska J. SHARPIN Regulates Uropod Detachment in Migrating Lymphocytes. Cell Rep. 2013 Nov 5. doi:pii: S2211-1247(13)00580-9.
Bouvard D†, Pouwels J†, De Francesci N, Ivaska J. Integrin inactivators: balancing cellular functions in vitro and in vivo. Nature Reviews Mol Cell Biol. Jul;14(7):430-42, 2013.
Pouwels J, Nevo J, Pellinen T, Ylänne J and Ivaska J. Negative regulators of integrin activity. J Cell Sci. Jul 15;125(Pt 14):3271-80, 2012.
Rantala JK†, Pouwels J†, Pellinen T, Veltel S, Mattila E, Laasola P, Potter CS, Duffy T, Sundberg JP, Kallioniemi O, Askari JA, Humphries M, Parsons M, Salmi M and Ivaska J. SHARPIN is an endogenous inhibitor of beta1-integrin activation. Nature Cell Biol. 13(11):1315-1324, 2011.
Hämälistö S†, Pouwels J†, De Franceschi N, Saari M, Ivarsson Y, Zimmermann P, Brech A, Stenmark H, Ivaska J. A ZO-1/?5?1-integrin complex regulates cytokinesis downstream of PKC? in NCI-H460 cells plated on fibronectin. PLOS ONE., 10.1371/journal.pone.0070696, 2013.
Salmela AL, Pouwels J, Mäki-Jouppila J, Kohonen P, Toivonen P, Kallio L, Kallio M. Novel pyrimidine-2,4-diamine derivative suppresses the cell viability and spindle assembly checkpoint activity by targeting Aurora kinases. Carcinogenesis. 2013 Feb;34(2):436-45.
Salmela AL†, Pouwels J†, Kukkonen-Macchi A, Waris S, Toivonen P, Jaakkola K, Mäki-Jouppila J, Kallio L, Kallio MJ. The flavonoid eupatorin inactivates the mitotic checkpoint leading to polyploidy and apoptosis. Exp Cell Res. 2012 Mar 10;318(5):578-92.
Kukkonen-Macchi A, Sicora O, Kaczynska K, Oetken-Lindholm C, Pouwels J, Laine L and Kallio MJ. Loss of p38? MAPK induces pleiotropic mitotic defects and massive cell death. J Cell Sci. 2011 Jan 15;124(Pt 2):216-27.
Salmela AL†, Pouwels J†, Varis A, Kukkonen AM, Toivonen P, Halonen PK, Perälä M, Kallioniemi O, Gorbsky GJ, Kallio MJ. Dietary flavonoid fisetin induces a forced exit from mitosis by targeting the mitotic spindle checkpoint. Carcinogenesis. 2009 Jun;30(6):1032-40.
Ahonen LJ, Kukkonen AM, Pouwels J, Bolton MA, Jingle CD, Stukenberg PT, Kallio MJ. Perturbation of Incenp function impedes anaphase chromatid movements and chromosomal passenger protein flux at centromeres. Chromosoma. 2009 Feb;118(1):71-84.
Pouwels J†, Kukkonen AM†, Lan W, Daum JR, Gorbsky GJ, Stukenberg T, Kallio MJ. Shugoshin 1 plays a central role in kinetochore assembly and is required for kinetochore targeting of Plk1. Cell Cycle. 2007 Jul 1;6(13):1579-85.