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. Author manuscript; available in PMC: 2014 Mar 10.
Published in final edited form as: Sci Signal. 2013 Sep 10;6(292):10.1126/scisignal.2004324. doi: 10.1126/scisignal.2004324

Fig. 4. JNK induces phosphorylation of Ajuba family proteins to increase binding to LATS1.

Fig. 4

A) Western blots on lysates of HEK293 cells co-transfected with Ajuba:V5, LIMD1:V5, or WTIP:V5 and, as indicated, MKK7B2:FLAG:JNK1a1 (activated JNK1), MKK7B2:FLAG:JNK1a1(APF) (inactive JNK1), or MKK7B2:FLAG:JNK2a2 (activated JNK2). The expression of transfected JNK constructs is shown in Flag blots. For each Ajuba family protein, the upper blot shows a standard gel, and the lower blot shows a Phos-tag gel. Blots are representative of 3 biological replicates. B) In vitro binding of LIMD1 to LATS1 after in vitro phosphorylation of LIMD1 by active JNK2. The upper blot shows amount of LIMD1 on beads, and the lower blot shows Myc:LATS1 bound to the beads. The histogram shows the average LATS1/LIMD1 ratio from three biological replicates, normalized to the ratio without JNK2 phosphorylation. C) In vitro binding assays comparing wild-type LIMD1 and LIMD12SA mutant binding to LATS1. Anti-V5 beads blot shows LIMD1, LIMD12SA or GFP (control) protein on beads. Co-IP shows Myc:LATS1 bound to wild-type or mutant LIMD1. Expression of constitutively active JNK2 is shown in LIMD1 Lysates blot. The histogram shows the average LATS1/LIMD1 ratio from three biological replicates, normalized to the ratio in wild-type LIMD1 control. Error bars in B,C show standard error. P values less than 0.05 are indicated. D) Model illustrating influence of JNK on Hippo signaling, active proteins are outlined in black, P indicates phosphorylation. In the absence of JNK activation, MST (Hpo), MOB (Mats), and WW45 (Sav) can activate LATS (Wts), which then represses YAP (Yki) by phosphorylating it. When JNK is active, it promotes phosphorylation of Ajuba family proteins (LIMD1, WTIP, or Jub), which then bind more strongly to LATS. This binding inhibits LATS phosphorylation and consequent activation of LATS, possibly by occluding the phosphorylation site, or by inhibiting binding of WW45 or MOB. E) Model illustrating the proposed conformational change of Ajuba family proteins caused by JNK that enhances LATS binding. Without JNK activation, Ajuba family proteins stay as a ‘closed’ form and cannot be accessed by LATS (left). When JNK is activated, the N-terminus of Ajuba family proteins get phosphorylated which result in exposure of C-terminus to LATS (right).