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. Author manuscript; available in PMC: 2008 Mar 1.
Published in final edited form as: Dev Cell. 2007 Mar;12(3):335–347. doi: 10.1016/j.devcel.2007.02.009

Figure 2. CKI ε phosphorylates SIPA1L1, promotes its degradation, and alleviates SIPA1L1-mediated Rap1 inhibition.

Figure 2

(A) CKIε expression causes an electrophoretic mobility shift of SIPA1L1(FL) but not SIPA1L1(CΔ). IB, Immunoblot of whole cell lysates from HEK 293 cells expressing the indicated proteins. CKIε and α-tubulin abundance are shown in the middle and bottom panels.

(B) SIPA1L1 is phosphorylated by CKIε. Myc-SIPA1L1 plus CKIε or GFP (as the negative control for IP) were expressed in HEK 293 cells. Myc-SIPA1L1 was precipitated and treated with or without CIP.

(C) CKIε stimulates SIPA1L1 degradation. Lanes 1–5, Myc-SIPA1L1 was co-expressed with CKIε(WT) or CKIε(KD) as indicated. Lane 5 and lane 6, cells expressing Myc-SIPA1L1 were treated with IC261 (50 μM) for 5 hr. IC261 treatment increased SIPA1L1 abundance (compare lane 5 to lane3 and lane 6 to lane 2). Expression of CKIε(WT/KD) and endogenous α-tubulin abundance are shown in middle and lower panels.

(D) CKIε rescues SIPA1L1-mediated Rap1 inhibition. Myc-SIPA1L1(FL) or Myc-SIPA1L1(CΔ) was co-expressed with or without CKIε in the CHO cells, followed by GST-RalGDS pull-down assay. The abundance of GTP-Rap1 and total Rap1 was quantified by ImageQuant. The ratio of GTP-Rap1 to total Rap1 is shown in the lower panel.