The kinetochore surveillance phosphatase PP2A-B56 dephosphorylates Knl1 to silence the spindle assembly checkpoint after all chromosomes have been correctly attached to microtubules.

Supplemental Figure legendsFigure S1. MPS1 autophosphorylation and localisation are regulated by PP2A-B56. (A) Control depleted or MPS1-depleted HeLa MPS1-GFP mitotic cells were treated with MPS1 inhibitor or DMSO for 5 min as indicated. Cells were stained with antibodies against MPS1 pT676 and CENP-C. Total MPS1-GFP was detected by GFP fluorescence. (B) Recombinant, insect cell expressed wild type (WT) or kinasedead (KD) His-MPS1 was treated with l-phosphatase or reaction buffer alone and analysed by SDS-PAGE and Coomassie Brilliant Blue staining (CBB) or Western blotting with antibodies against MPS1 pT676. (C) MPS1 T-loop phosphorylation and endogenous MPS1 levels were assessed in control depleted mitotic HeLa MPS1-GFP cells or HeLa MPS1-GFP cells depleted of PP1αβγ (siPP1), PP2CA (siPP2A), PPP4C (siPP4), PPP5C (siPP5) or PPP6C (siPP6) and treated with MPS1i as indicated. Mean kinetochore MPS1 pT676 phosphorylation (D) and MPS1-GFP kinetochore levels ± SEM (E) relative to CENP-C are plotted. Values are from three independent experiments with 15 kinetochores measured in at least 10 cells per condition. (F) Representative Western blot analysis of PP1, PP2A, PP4, PP5 and PP6 depletion efficiencies. Actin is shown as a loading control. (G) Representative Western blot of PP2A-B55 and PP2A-B56 depletion efficiencies. Actin is used as a loading control. (H) Western blot of HeLa-Flp-In/TREx GFP-BUBR1 WT or GFP-

Inflammatory skin conditions are increasingly recognised as being associated with systemic inflammation. The mechanisms connecting the cutaneous and systemic disease are not well understood. CD1a is a virtually monomorphic major histocompatibility complex (MHC) class I-like molecule, highly expressed by skin and mucosal Langerhans cells, and presents lipid antigens to T-cells. Here we show an important role for CD1a in linking cutaneous and systemic inflammation in two experimental disease models. In human CD1a transgenic mice, the toll-like receptor (TLR)7 agonist imiquimod induces more pronounced splenomegaly, expansion of the peripheral blood and spleen T cell compartments, and enhanced neutrophil and eosinophil responses compared to the wild-type, accompanied by elevated skin and plasma cytokine levels, including IL-23, IL-1α, IL-1β, MCP-1 and IL-17A. Similar systemic escalation is shown in MC903-induced skin inflammation. The exacerbated inflammation could be counter-acted by CD1a-blocking antibodies, developed and screened in our laboratories. The beneficial effect is epitope dependent, and we further characterise the five best-performing antibodies for their capacity to modulate CD1a-expressing cells and ameliorate CD1a-dependent systemic inflammatory responses. In summary, we show that a therapeutically targetable CD1a-dependent pathway may play a role in the systemic spread of cutaneous inflammation.

During mitosis, the formation of microtubule-kinetochore attachments is monitored by the serine/threonine kinase Mono-Polar Spindle 1 (MPS1).MPS1 is recruited to unattached kinetochores where it phosphorylates KNL1, BUB1 and MAD1 to initiate the spindle checkpoint. This arrests the cell cycle until all kinetochores have been stably captured by microtubules. MPS1 also contributes to the error correction process rectifying incorrect kinetochore attachments. MPS1 activity at kinetochores requires auto-phosphorylation at multiple sites including T676 in the activation segment or "T-loop". We now demonstrate that a BUBR1-bound pool of PP2A-B56 regulates MPS1 T-loop autophosphorylation and hence activation status in mammalian cells.Overriding this regulation using phospho-mimetic mutations in the MPS1 Tloop to generate a constitutively active kinase results in a prolonged mitotic arrest with continuous turn-over of microtubule-kinetochore attachments.Dynamic regulation of MPS1 catalytic activity by kinetochore-localized PP2A-B56 is thus critical for controlled MPS1 activity and timely cell cycle progression.