The transmembrane protein CBP plays a role in transiently anchoring small clusters of Thy-1, a GPI-anchored protein, to the cytoskeleton

Abstract

It remains unclear how GPI-anchored proteins (GPIAPs), which lack cytoplasmic domains, transduce signals triggered by specific ligation. Such signal transduction has been speculated to require the ligated GPIAP to associate with membrane-spanning proteins that communicate with obligate cytoplasmic proteins. Transient anchorage of crosslinked proteins on the cell surface was previously characterized by single-particle tracking, and temporary association with the actin cytoskeleton was hypothesized to cause regulated anchorage. GPIAPs, such as Thy-1, require clustering, cholesterol and Src-family kinase (SFK) activity to become transiently anchored. By contrast, a transmembrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), which has a PDZ-binding motif in its cytoplasmic C-terminus that binds the ERM adaptor EBP50, exhibits anchorage that strictly requires EBP50 but has little dependence on cholesterol or SFK. We hypothesized that a transmembrane protein would be required to mediate the linkage between Thy-1 and the cytoskeleton. Here, we present evidence, obtained by shRNA knockdown, that the transmembrane protein Csk-binding protein (CBP) plays an obligatory role in the transient anchorage of Thy1. Furthermore, either a dominant-negative form of CBP that did not bind EBP50 or a dominant-negative EBP50 drastically reduced transient anchorage of Thy-1, indicating the involvement of this adaptor. Finally, we speculate on the role of phosphorylation in the regulation of transient anchorage.

Fig. 1.

Single-particle tracking (SPT) using quantum dots (Qdots) gives comparable results to gold-particle-based SPT. (A) TA of Thy-1 clusters labeled by Qdots (white bars) in C3H cells was induced by (B, left) the maximal crosslinking [crslk; see procedure 1 in Materials and Methods; the number of trajectories (n)=70] scheme or by (B, right) preassembled complexes (prcmlx; see procedure 2 in Materials and Methods; n=78), whereas the control observation was made in cells treated without crosslinking tertiary antibody (ctrl; n=60). The Qdot-based SPT results are similar to the results previously published for gold-particle-based SPT (hatched bars; n=69 for ctrl, n=90 for crslk and n=92 for prcmplx) (Chen et al., 2006). (C,D) Thy-1-cluster-bound Qdots underwent TA ranging from ∼700 ms to more than 7 seconds (C) and exhibited a bi-exponential decay in TA duration distribution, similar to the results previously obtained in gold-particle-based SPT (D).

Fig. 2.

The transmembrane protein CBP mediates TA of Thy-1. (A, left) After shRNA for CBP was introduced in 3T3 cells, TA of Thy-1 clusters was markedly reduced (n=57) compared with control 3T3 cells (n=40) or cells transfected with a random DNA sequence (n=79). Without CBP, there was minimal TA after pervanadate treatment (n=33). After CBP shRNA was introduced, 3T3 cells were infected with retroviral viruses coding for either full-length CBP (CBPFL, n=49) or a CBP construct with the TRL deletion (CBPDTRL, n=55), which does not bind EBP50 (right). (A, right) Compared with CBP knockdown cells, TA was partially rescued by full-length CBP, whereas almost no rescue was achieved with the TRL mutant. The negative control was mock transfection in the CBP-knockdown cells (n=30). (B) Blots show that CBP was knocked down in whole-cell lysates (WCL) and the effective restoration of CBPFL and CBPTRL in the shRNA-infected cells compared with the mock transfection. Molecular masses are indicated in kDa. (C) TA of Thy-1 clusters on C3H was prolonged by pervanadate treatment to over 1 minute. Every Thy-1-cluster-bound Qdot (red) was seen to be accompanied by proximate CBP staining (green). The inset in the upper left corner is the magnified view of the area surrounded by the white line. (D) In control C3H cells, in which not all Thy-1 clusters would be expected to be undergoing TA at the time of fixation, CBP staining (green) was not necessarily observed close to every Thy-1-cluster-bound Qdot (red). Scale bars: 5 μm.

Fig. 3.

The cytoskeletal adaptor, EBP50, is required for TA of both the GPIAP Thy-1 and the transmembrane protein CFTR. C3H cells were transfected with GFP-tagged wild-type EBP50 (EGFP–WT-EBP50, labeled as wt-EBP50) or dominant-negative EBP (EGFP–DN-EBP50, labeled as dn-EBP50), which fails to bind to ERM proteins. SPT using preassembled Qdot complexes showed that cells with overexpressed EGFP–DN-EBP50 had significantly reduced TA for both Thy-1 clusters (n=64) and CFTR molecules (n=52) compared with the control cells (n=78) and cells transfected with EGFP–WT-EBP50 (n=58). CFTR-bound Qdots were immobile when EGFP–DN-EBP was not present (see supplementary material Movie 2). This is depicted as 100% relative anchorage time.

Fig. 4.

EBP50-ERM assembly is the common adaptor complex for both Thy-1 clusters and CFTR. (A) Thy-1 clusters are generated by tertiary complexes consisting of streptavidin-coated Qdots binding biotinylated mouse anti-IgG, which binds anti-Thy-1 mAb. CBP, alone or complexed with other unknown Src-family kinase substrates (KS), is either recruited into Thy-1 clusters or captured by Thy-1 clusters. Here, CBP and/or KS are phosphorylated by SFKs, enabling CBP to bind to actin filaments via the EBP50-ERM assembly. The anchorage can be terminated when either CBP or the adaptors are dephosphorylated by an unspecified phosphatase (PTP). (B) CFTR, containing its own PDZ-binding motif, binds to actin filaments via EBP50-ERM spontaneously without extensive crosslinking. This anchorage is not dependent on SFK phosphorylation.