The Sharpin interactome reveals a role for Sharpin in lamellipodium formation via the Arp2/3 complex

Khan, Meraj Hasan; Salomaa, Siiri I.; Jacquemet, Guillaume; Butt, Umar; Miihkinen, Mitro; Deguchi, Takahiro; Kremneva, Elena; Lappalainen, Pekka; Humphries, Martin J.; Pouwels, Jeroen

doi:10.1242/jcs.200329

Cited by 16 publications

(19 citation statements)

References 66 publications

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“…Although the mechanism for this effect remains unknown, platelets can scavenge pathogen-derived peptides as well as self-peptides and load these onto MHC class I for cross-presentation to CD8 and Treg cells (27,41), thereby impacting immunity. SHARPIN, through its association with cytoskeletal protein Arp2/3 or with proteins involved in endosomal trafficking (42), may affect MHC class I delivery to the platelet plasma membrane or its re-endocytosis.…”

Section: Discussionmentioning

confidence: 99%

SHARPIN at the nexus of integrin, immune, and inflammatory signaling in human platelets

Kasirer-Friede

Tjahjono

Eto

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Platelets mediate primary hemostasis, and recent work has emphasized platelet participation in immunity and inflammation. The function of the platelet-specific integrin αIIbβ3 as a fibrinogen receptor in hemostasis is well defined, but the roles of αIIbβ3 or integrin-associated proteins in nonhemostatic platelet functions are poorly understood. Here we show that human platelets express the integrin-associated protein SHARPIN with functional consequences. In leukocytes, SHARPIN interacts with integrin α cytoplasmic tails, and it is also an obligate member of the linear ubiquitin chain assembly complex (LUBAC), which mediates Met1 linear ubiquitination of proteins leading to canonical NF-κB activation. SHARPIN interacted with αIIb in pull-down and coimmunoprecipitation assays. SHARPIN was partially localized, as was αIIbβ3, at platelet edges, and thrombin stimulation induced more central SHARPIN localization. SHARPIN also coimmunoprecipitated from platelets with the two other proteins comprising LUBAC, the E3 ligase HOIP and HOIL-1. Platelet stimulation with thrombin or inflammatory agonists, including lipopolysaccharide or soluble CD40 ligand (sCD40L), induced Met1 linear ubiquitination of the NF-κB pathway protein NEMO and serine-536 phosphorylation of the p65 RelA subunit of NF-κB. In human megakaryocytes and/or platelets derived from induced pluripotent stem (iPS) cells, SHARPIN knockdown caused increased basal and agonist-induced fibrinogen binding to αIIbβ3 as well as reduced Met1 ubiquitination and RelA phosphorylation. Moreover, these SHARPIN knockdown cells exhibited increased surface expression of MHC class I molecules and increased release of sCD40L. These results establish that SHARPIN functions in the human megakaryocyte/platelet lineage through protein interactions at the nexus of integrin and immune/inflammatory signaling.

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Section: Discussionmentioning

confidence: 99%

SHARPIN at the nexus of integrin, immune, and inflammatory signaling in human platelets

Kasirer-Friede

Tjahjono

Eto

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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“…16 Thus, SHANK3-mediated regulation of actin dynamics is required for normal neuronal development and function. SHANK3 associates with different actin regulators, including ABI1, 17 Abp1, 18,19 a-fodrin, 20 SHARPIN, 21 bPIX, 22 CaMKKII, 23,24 IRSp53, 25 and cortactin, 26,27 and SHANK3 mutations, identified in patients with ASD, impair SHANK3 association with actin in cells. 28 However, SHANK3 has not been reported to interact directly with actin, and the molecular mechanisms regulating the actin scaffolding functions of SHANK3 remain unknown.…”

Section: Introductionmentioning

confidence: 99%

SHANK3 conformation regulates direct actin binding and crosstalk with Rap1 signaling

et al. 2021

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Actin-rich cellular protrusions direct versatile biological processes from cancer cell invasion to dendritic spine development. The stability, morphology, and specific biological functions of these protrusions are regulated by crosstalk between three main signaling axes: integrins, actin regulators, and small guanosine triphosphatases (GTPases). SHANK3 is a multifunctional scaffold protein, interacting with several actin-binding proteins and a well-established autism risk gene. Recently, SHANK3 was demonstrated to sequester integrin-activating small GTPases Rap1 and R-Ras to inhibit integrin activity via its Shank/ProSAP N-terminal (SPN) domain. Here, we demonstrate that, in addition to scaffolding actin regulators and actin-binding proteins, SHANK3 interacts directly with actin through its SPN domain. Molecular simulations and targeted mutagenesis of the SPN-ankyrin repeat region (ARR) interface reveal that actin binding is inhibited by an intramolecular closed conformation of SHANK3, where the adjacent ARR domain covers the actin-binding interface of the SPN domain. Actin and Rap1 compete with each other for binding to SHANK3, and mutation of SHANK3, resulting in reduced actin binding, augments inhibition of Rap1-mediated integrin activity. This dynamic crosstalk has functional implications for cell morphology and integrin activity in cancer cells. In addition, SHANK3-actin interaction regulates dendritic spine morphology in neurons and autism-linked phenotypes in vivo.

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“…In neurons, SHANK3 associates with different actin regulators including Abi1 18,19 , Abp1 20 , α-fodrin 21 , SHARPIN 22 , βPIX 23 , CaMKKIIα 24 , IRSp53 25 , and cortactin 26,27 . In the context of ASD, SHANK3 mutations contribute to disease pathogenesis through dysregulation of the actin cytoskeleton 2,8,[28][29][30] and ASD symptoms of Shank3-deficient mice are alleviated by targeting actin regulators 30 .…”

Section: Introductionmentioning

confidence: 99%

Conformational dynamics regulate SHANK3 actin and Rap1 binding

Salomaa

Miihkinen

Kremneva

et al. 2020

Preprint

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Actin-rich cellular protrusions direct versatile biological processes from cancer cell invasion to dendritic spine development. The stability, morphology and specific biological function of these protrusions are regulated by crosstalk between three main signaling axes: integrins, actin regulators and small GTPases. SHANK3 is a multifunctional scaffold protein, interacting with several actin-binding proteins, and a well-established autism risk gene. Recently, SHANK3 was demonstrated to sequester integrin-activating small GTPases Rap1 and R-Ras to inhibit integrin activity via its N-terminal SPN domain. Here, we demonstrate that SHANK3 interacts directly with actin using its SPN domain. Actin binding can be inhibited by an intramolecular closed conformation of SHANK3, where the adjacent ARR domain covers the actin-binding interface of the SPN domain. Actin and Rap1 compete with each other for binding to SHANK3 and loss of SHANK3-actin binding augments inhibition of Rap1-mediated integrin activity. This dynamic crosstalk has functional implications for filopodia formation in cancer cells, dendritic spine morphology in neurons and autism-linked phenotypes in vivo.

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The Sharpin interactome reveals a role for Sharpin in lamellipodium formation via the Arp2/3 complex

Cited by 16 publications

References 66 publications

SHARPIN at the nexus of integrin, immune, and inflammatory signaling in human platelets

SHARPIN at the nexus of integrin, immune, and inflammatory signaling in human platelets

SHANK3 conformation regulates direct actin binding and crosstalk with Rap1 signaling

Conformational dynamics regulate SHANK3 actin and Rap1 binding

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