Inside-out regulation of L1 conformation, integrin binding, proteolysis, and concomitant cell migration

Abstract

Previous reports on the expression of the cell adhesion molecule L1 in pancreatic ductal adenocarcinoma (PDAC) cells range from absent to high. Our data demonstrate that L1 is expressed in poorly differentiated PDAC cells in situ and that threonine-1172 (T1172) in the L1 cytoplasmic domain exhibits steady-state saturated phosphorylation in PDAC cells in vitro and in situ. In vitro studies support roles for casein kinase II and PKC in this modification, consistent with our prior studies using recombinant proteins. Importantly, T1172 phosphorylation drives, or is associated with, a change in the extracellular structure of L1, consistent with a potential role in regulating the shift between the closed conformation and the open, multimerized conformation of L1. We further demonstrate that these distinct conformations exhibit differential binding to integrins alphavbeta3 and alphavbeta5 and that T1172 regulates cell migration in a matrix-specific manner and is required for a disintegrin and metalloproteinase-mediated shedding of the L1 ectodomain that has been shown to regulate cell migration. These data define a specific role for T1172 of L1 in regulating aspects of pancreatic adenocarcinoma cell phenotype and suggest the need for further studies to elucidate the specific ramifications of L1 expression and T1172 phosphorylation in the pathobiology of pancreatic cancer.

Figure 1.

αL1 mAb 2C2 does not detect L1 expressed by PDAC cells in situ. Sections were stained with UJ127 (A, C, and E) or 2C2 (B, D, and F) (brown). Very poorly differentiated (anaplastic) PDAC cells are UJ127 positive (A), 2C2-negative (B). Poorly differentiated (G3) cells of a malignant duct are UJ127 positive (C), 2C2 negative (D). Nerve bundles from the same section as A and B are UJ127 positive (E), 2C2 positive (F).

Figure 2.

Mapping the 2C2 epitope. (A) Immunoblot of the indicated cells with 2C2, αECD, and αErk2. (B) ELISA of GST proteins encoding the indicated sequences with 2C2 or αGST. (C) Cells were treated with SS and immunoblotted with 2C2, αECD, and αErk2. (D) M21 cells treated with CalA were immunoblotted with 2C2, αECD, and αErk2. The blot was reprobed with the αP-T1172 pAb. (E) Cells were treated for 90 min with the indicated concentrations of OA and immunoblotted with 2C2, αECD, and αErk2.

Figure 3.

PKC is involved in regulating 2C2 epitope availability but is incapable of phosphorylating T1172 of recombinant L1 CD in isolation. (A and B) Cells were treated with the PKC inhibitors CalC (A) or BisI (B) and immunoblotted with 2C2, αECD, and αErk2. (C) Cells were treated with CalC for 90 min before washout and replenishing of basal media or media containing the PKC-activating phorbol ester PMA. Lysates were immunoblotted with 2C2 and αECD. (D) RT-PCR with PKC isoform-specific primers. GAPDH, control. (E) GST-L1 nonneuronal CD (L1) or histone-H1 were incubated with PKC isoforms. T1172 phosphorylation and H1 phosphorylation were detected with αP-T1172 and αP-S/T-F pAbs, respectively.

Figure 4.

CKII phosphorylates T1172 of the L1 CD and phosphorylation of T1172 is responsible for loss of 2C2 signal. (A) Cells treated with the CKII inhibitor DMAT were immunoblotted with 2C2, αECD, and αErk2. (B and C) GST-L1 CD proteins were incubated with CKII. T1172 phosphorylation was detected with αP-T1172 (B) or 2C2 (C). T1172A, phosphorylation control. Neuronal protein integrity was verified by assessing S1181 phosphorylation with αP-S1181 (B). (D) L1 from CalA-treated Panc1 cell lysates was captured on immobilized anti-L1 antibodies before treatment with alkaline phosphatase with or without subsequent treatment with CKII as described in Materials and Methods. Bound L1 was then detected with 2C2 or the αP-T1172 pAb.

Figure 5.

Cytoplasmic S/T phosphorylation promotes or accompanies changes in the L1 ECD conformation. (Ai) L1 was IPed from Panc1 cells by using the αP-T1172 or T1172-independent (T1172-IND) pAb. (Aii) L1 was IPed from untreated or CalA-treated M21 cells with the αP-T1172 pAb. (B) Panc1 (Bi) or CHO cells stably expressing wild-type noneuronal L1 or L1 containing a T1172A mutation (Bii) were treated with CalA or SS and processed for IP with 5G3 and UJ127. IPs were immunoblotted with αECD. (C and D) Panc1 cells were treated with CalA or SS and processed for FACS with UJ127 (Ci), 5G3 (Cii), Neuro4 (Ciii), αECD (D, i and ii), or αFL (D, i and ii). FACS controls received secondary alone (solid).

Figure 6.

ECD conformation regulates the availability of αL1 antibody epitopes. Immobilized GST proteins encoding the indicated domains were incubated with the indicated antibodies. (A) Neuro4 or 5G3 detection of Ig domains. Inset, reducing immunoblot of Ig1-3 with 5G3, Neuro4, or αHis. (B) Competition assay: Ig1-2 was incubated with or without 10-fold excess of Neuro4 or 5G3 and then probed with the opposite antibody and isotype-specific secondary antibody. (C) Domain mapping of αFL and αECD. (Ci) Relative binding of the αECD plotted as percent of αFL binding to the indicated fusion proteins. (Cii) Relative binding of each pAb to the indicated fusion proteins. (D) Multimerization analysis: wild-type FN3 domain (WT) or FN3 domain harboring the dibasic C-C′ loop mutation (FN3 mut) that impairs multimerization (Silletti et al., 2000a) were probed with αFL or αECD. (E) Sequential domain addition to map Ig domain interactions using αFL or αECD.

Figure 7.

Regulation of L1 proteolysis and integrin-binding by CD phosphorylation and ECD conformation. (A and B) Panc1 cells were treated with CalA, OA, or SS in the presence or absence of TAPI1 (TAPI). Conditioned media and cell lysate were immunoblotted with αECD or αactin. (C) Panc1 cells were treated with DMAT or BisI in the presence or absence of PMA. Conditioned media and cell lysate were immunoblotted with αECD or αactin. (D) CHO-K1 cells stably expressing wild type (WT) or T1172A mutant (T1172A) nonneuronal L1 were treated with CalA and immunoblotted with αECD or αactin. (E) Solid phase integrin capture assay of αvβ3 and αvβ5 binding to soluble L1-ECD, FN3 domain (FN3-His), or biotinylated vitronectin (biotinVN) as detected with 5G3, αFL, αECD, αHis, or αbiotin (αBio). (F and G) LamininI or fibronectin haptotactic migration (F) or adhesion (G) of mock-transfected (Mock) or CHO-K1 cells stably expressing wild-type nonneuronal (WT) or T1172A mutant (T1172A) nonneuronal L1.

Figure 8.

L1 ectodomain regulation and cytoplasmic phosphorylation. (A) Putative model of L1 ectodomain conformation and its regulation by, or association with, changes in intracellular phosphorylation state. Availability of epitopes and interactions with integrins and proteases are shown. (B) Aggregation of J558L-L1 myeloma cells in the presence or absence of 50 μg/ml 5G3 or Neuro4.