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First published online May 28, 2004
doi: 10.1242/10.1242/jcs.01139

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Cytoskeletal restraints regulate homotypic ALCAM-mediated adhesion through PKC independently of Rho-like GTPases

Aukje W. Zimmerman^1,*, Judith M. D. T. Nelissen^1,*, Sjenet E. van Emst-de Vries², Peter H. G. M. Willems², Frank de Lange¹, John G. Collard³, Frank N. van Leeuwen¹ and Carl G. Figdor^1,

¹ Department of Tumor Immunology, University Medical Center St Radboud, Nijmegen, The Netherlands
² Department of Biochemistry, University Medical Center St Radboud, Nijmegen, The Netherlands
³ Division of Cell Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands

View larger version (18K): [in a new window]   Fig. 1. (A) Flow-cytometric analysis of ALCAM expression by KG1 and K562-ALCAM cells. The shaded histograms represent the isotype control, whereas the white histograms represent ALCAM staining with AZN-L50. (B) ALCAM-mediated adhesion of both K562-ALCAM (white bars) and KG1 cells (black bars) to immobilized ALCAM-Fc requires disruption of the actin cytoskeleton. The cells were pre-incubated for 30 minutes at 37°C with or without CytD (2.5 µg/ml) or LatA (5 µg/ml) and subsequently allowed to adhere to an ALCAM-Fc coated plate (250 ng/ml ALCAM-Fc) for 45 minutes at 37°C in the presence or absence of the ALCAM-blocking mAb AZN-L50. Non-ALCAM antibodies did not affect binding, showing that the observed adhesion is ALCAM-mediated (data not shown). Specific adhesion is expressed as the mean percentage (±s.d.) of adherent cells from triplicate wells after subtraction of the adhesion in the presence of the blocking mAb AZN-L50. Data are representative of three experiments. (C) CytD treatment does not alter the affinity of K562-ALCAM (circles) and KG1 cells (triangles) for ALCAM-Fc. Cells were treated with (open symbols) or without (closed symbols) CytD (as in B) and subsequently incubated with the indicated dilutions of ALCAM-Fc. The percentage of cells that have bound ALCAM-Fc is detected by flow cytometry using a FITC-conjugated secondary goat-anti-human-Fc antibody.

View larger version (50K): [in a new window]   Fig. 2. CytD and LatA transiently activate RhoA, Rac1 and Cdc42. K562-ALCAM cells (A) and KG1 cells (B) in suspension were treated with 2.5 µg/ml CytD for the indicated time. Subsequently, cell lysates were incubated with GST-PAK-CD or GST-C21 and the bound RhoA, Rac1 and Cdc42 molecules were detected by western blot with the respective antibodies. Total lysates served as a control for analyzing total amounts of RhoA, Rac1 or Cdc42 on western blot using the respective antibodies. Similar observations were made using parental K562 cells (data not shown).

View larger version (32K): [in a new window]   Fig. 3. (A) Expression of Myc-tagged Rho-like GTPase-mutants and HA-tagged C1199-Tiam1 in KG1. KG1 cells were retrovirally transduced with dominant negative (N19-RhoA, N17-Rac1, N17-Cdc42) or constitutively active (V14-RhoA, V12-Rac1, V12-Cdc42, L61-Rac1) GTPase mutants, or with C1199-Tiam1. Cells were lysed and expression of the mutants was detected by western blotting using anti-Myc and anti-HA antibodies, respectively. C1199-Tiam1 and the Rho-like GTPase mutants migrated according to the expected heights (RhoA > Rac1=Cdc42). (B) Rho-like GTPase mutants and C1199-Tiam1 do not alter adhesion of KG1 cells. Cells expressing either dominant negative GTPase (N19-RhoA, N17-Rac1, N17-Cdc42), constitutively active GTPase (V14-RhoA, V12-Rac1, V12-Cdc42, L61-Rac1) or C1199-Tiam1 were allowed to adhere to ALCAM-Fc-coated beads after pre-incubation with (white bars) or without (black bars) mAb AZN-L50, for 45 minutes at 37°C. Adhesion is expressed as the percentage of bound cells. Data are representative of three independent experiments.

View larger version (35K): [in a new window]   Fig. 4. (A) Inhibition of PKC dose-dependently reduced CytD-induced adhesion. KG1 cells were pre-treated for 10 minutes at 37°C with increasing concentrations of either chelerythrine chloride (black bars) or myristoylated PKC inhibitor (white bars) in combination with CytD (2.5 µg/ml). Both inhibitors of PKC significantly reduce CytD-induced adhesion to immobilized ALCAM. (B) PKC requirement is not restricted to CytD but applies to LatA as well. KG1 cells were pre-treated with chelerythrine chloride (Chel, 5 µM) or myristoylated PKC inhibitor (Myr, 100 µM) for 10 minutes at 37°C in combination with either CytD (2.5 µg/ml, black bars) or LatA (5 µg/ml, gray bars). Induction of adhesion by both cytoskeleton-disrupting drugs is equally inhibited by both chelerythrine chloride and myristoylated PKC inhibitor, demonstrating that the requirement for PKC is not restricted to the mode of action of CytD in itself. Similar observations were made for K562-ALCAM (not shown). (C) Analysis of the distribution of ALCAM and actin by CLSM. KG1 cells were pre-treated without (a) or with CytD (2.5 µg/ml, b), or with a combination of CytD and chelerythine chloride (Chel, 5 µM, c). Cells were stained with mAb AZN-L50 (green) and Texas Red-X phalloidin (red). For each preparation the same instrument settings were used. The scale bar represents 10 µm. Similar results were obtained with K562-ALCAM cells (not shown). (D) PKC requirement is not restricted to homotypic ALCAM-mediated adhesion induced by cytoskeleton-disrupting agents, but also applies to heterotypic ALCAM-CD6 adhesion. KG1 cells were pre-treated with PKC inhibitors as described in B, in the presence or absence of CytD (2.5 µg/ml). Cells were allowed to adhere to a plate coated with 250 ng/ml of CD6-Fc (white bars) or ALCAM-Fc (black bars) for 45 minutes at 37°C. Both spontaneous and CytD-induced ALCAM-CD6 adhesion are inhibite d by chelerithrine chloride and myristoylated PKC inhibitor. Similar observations were made for K562-ALCAM cells (not shown).

View larger version (19K): [in a new window]   Fig. 5. (A) Long-term PMA treatment leads to downregulation of PKC and PKC. KG1 and K562-ALCAM cells were cultured for 24 hours in presence or absence of PMA (50 nM) to induce downregulation of PKC. PKC expression was analyzed by western blotting. PKC isotypes and are both downregulated upon culturing in the presence of PMA in both cell lines. Expression of PKC isotypes and was not affected (data not shown). (B) PMA culturing inhibits cytoskeleton-dependent ALCAM-mediated adhesion. KG1 cells were cultured for 16 hours in the presence (white bars) or absence (black bars) of 50 nM PMA. Subsequently, induction of adhesion by pre-incubation of the cells for 30 minutes at 37°C with CytD (2.5 µg/ml) or LatA (5 µg/ml) was determined, in the presence or absence of the blocking antibody AZN-L50 (10 µg/ml). Downregulation of PKC upon culturing with PMA inhibits CytD- and LatA-induced adhesion to background levels. Similar observations were made for K562-ALCAM (not shown). (C) PMA culturing does not affect the overall ALCAM expression. ALCAM expression on KG1 cultured in the presence or absence of PMA (50 nM) was determined by flow cytometry. Shaded histograms represent isotype control staining and white histograms represent ALCAM staining with mAb AZN-L50.

View larger version (22K): [in a new window]   Fig. 6. Effect of PKC downregulation or PKC inhibition on ALCAM-affinity. Soluble ALCAM-Fc-binding assay with KG1 cells (A) and K562-ALCAM (B) either cultured for 16 hours with () or without () 50 nM PMA or pre-incubated for 10 minutes at 37°C with chelerythrine chloride (5 µM, ) or myristoylated PKC pseudosubstrate (100 µM, ). Cells are incubated with increasing concentrations of soluble ALCAM-Fc and the percentage of cells that have bound ligand is determined by flow cytometry. Data are representative of three independent experiments.

View larger version (25K): [in a new window]   Fig. 7. (A) Surface expression of ALCAM on K562-ALCAMSer, K562-ALCAMThr and wild-type K562-ALCAM. K562 cells were transfected with expression constructs coding for mutant ALCAM proteins and were sorted to obtain homogeneous cell populations expressing similar levels of mutant ALCAM. Expression was analyzed by flow cytometry. The shaded histograms represent isotype control staining and the white histograms represent staining with ALCAM-antibody AZN-L50. (B) Replacement of serine- or threonine residues in the cytoplasmic domain of ALCAM does not affect spontaneous or CytD-induced adhesion. Adhesion of K562-ALCAMSer, K562-ALCAMThr and K562-ALCAM was analyzed after treatment of cells with or without CytD (2.5 µg/ml) as in Fig. 1B. Wild-type and mutant ALCAM-expressing cells show similar patterns of adhesion. Data are representative of three experiments. (C) No evidence for phosphorylation of ALCAM after treatment with [32P]-phosphate. K562 and K562-ALCAM cells were incubated overnight with [35S]methionine/cysteine (a) or for 3 hours with [32P]-disodiumphosphate (b). Cells were pre-incubated with 50 nM PMA for 16 hours prior to [32P]-phosphate labeling (PKC downregulation), or 15 minutes after [32P]-phosphate labeling (PKC activation), respectively. ALCAM was immunoprecipitated from labeled cell lysates with 1 µg of AZN-L51. ALCAM was detected in [35S]methionine/cysteine labeled K562-ALCAM lysate, but no [32P]phosphate-labeled ALCAM could be detected, not even after PMA stimulation. Long-term PMA treatment resulted in decreased overall protein phosphorylation. Similar observations were made with KG1 cells (not shown).

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