Model of potential NDR activation at the membrane. NDR is recruited by hMOB to the plasma membrane, thereby bringing NDR into the proximity of its upstream kinase (HMK [hydrophobic motif kinase]). Subsequently, NDR is phosphorylated on Thr444 by HMK, while Ser281 is phosphorylated by NDR itself, leading to full activation of NDR at the plasma membrane. The active kinase is inactivated by PP2A. The HMK is apparently also regulated by PP2A (Stegert et al., submitted for publication).

Membrane-targeted hMOB1A stabilizes native NDR1 proteins. A. U2-OS cells were transfected with empty vector, HA-hMOB1A, or mp-HA-hMOB1A before analysis by immunoblotting using anti-T444-P (top panel), anti-NDR1 (middle top panel), anti-HA 12CA5 (middle bottom panel), and anti-α-tubulin (bottom panel) antibodies. Arrowheads indicate NDR1 forms. B. U2-OS cells transfected with empty vector, HA-hMOB1A, or mp-HA-hMOB1A were subjected to S100/P100 fractionation (S, cytoplasm; P, membrane) before immunoblotting with anti-T444-P (top panel), anti-NDR1 (middle panel), and anti-HA 12CA5 (bottom panel) antibodies. C. U2-OS cells expressing wild-type (top panels) or membrane-targeted hMOB1A (bottom panels) were analyzed by indirect immunofluorescence with anti-T444-P (green) and anti-HA 12CA5 (red) antibodies. Nuclei are in blue.

NDR1 phosphorylated on Thr444 is mostly cytoplasmic. A. COS-7 cells expressing HA-tagged wild-type (wt) (top panels), Thr444Ala (T444A) (middle panels), or kinase-dead (K118A) (bottom panels) NDR1 were either left untreated (− OA) or incubated with okadaic acid (+ OA) before processing for immunofluorescence using anti-HA 12CA5 (red) and anti-T444-P (green) antibody. Nuclei are indicated in blue. All pictures were taken using the same settings, which had been adjusted to signals of cells overexpressing NDR1. B. COS-7 cells expressing HA-tagged NDR1 were subjected to biochemical cell fractionation after OA treatment (T, total; N, nuclear; C, cytoplasmic; M, membrane) and processed for immunoblotting with anti-T444-P (top panel), anti-HA 12CA5 (top middle panel), anti-lamin A/C (middle panel), anti-α-tubulin (bottom middle panel), and anti-p63/Climp (bottom panel) antibodies. C. After OA treatment, U2-OS cells were subjected to biochemical cell fractionation (T, total; N, nuclear; C, cytoplasmic; M, membrane) and processed for immunoblotting with anti-T444-P (top panel), anti-NDR1 (top middle panel), anti-lamin A/C (middle panel), anti-α-tubulin (bottom middle panel), and anti-p63/Climp (bottom panel) antibodies.

hMOB1A is mostly cytoplasmic, in contrast to hMOB2; however, both species colocalize with NDR1 in the cytoplasm/membrane. A. COS-7 cells expressing either GFP-hMOB1A (top panels), myc-hMOB1A (second top panels), HA-hMOB1A (middle top panels), Flag-hMOB2 (middle bottom panels), myc-hMOB2 (second bottom panels), or HA-hMOB2 (bottom panels) were either left untreated (− OA) or incubated with 1 μM okadaic acid (+ OA), before processing for immunofluorescence microscopy using no, anti-myc, anti-HA Y11, or anti-Flag M2 antibody. GFP/FITC signals are shown in green. Nuclei are indicated in blue. B. Cells cotransfected with myc-hMOB1A/HA-NDR1 (top panels) or myc-hMOB2/HA-NDR1 (bottom panels) were analyzed by immunofluorescence microscopy using anti-myc 9E10 (green) and anti-HA Y11 (red) antibodies. Merged images of hMOB and NDR1 staining are shown (merge). Enlargement of the region indicated by a white rectangle is shown in the middle panels. Arrowheads indicate colocalization sites at the plasma membrane.

Activation of nucleus-targeted NDR1 results in cytoplasmic accumulation of NDR species. A. Lysates of COS-7 cells expressing HA-tagged wild-type (wt), SV40NLS-tagged (NLS) wt, or kinase-dead (kd) NDR1 were analyzed by immunoprecipitation (IP) using anti-HA 12CA5 antibody. Complexes were assayed by Western blotting using anti-T444-P (top panel), anti-S281-P (middle panel), and anti-HA (bottom panel) antibodies. Prior to lysis, cells were incubated with (+) or without (−) OA. B. In parallel, immunocomplexes were subjected to kinase assays. Error bars indicate standard deviations. C. COS-7 cells expressing SV40NLS-tagged wild-type (top panels), kinase-dead (K118A) (top middle panels), Thr444Ala (T444A) (bottom middle panels), or MOB1A-binding-deficient (Y31A) (bottom panels) NDR1 were either left untreated (− OA) or incubated with okadaic acid (+ OA) before processing for immunofluorescence using anti-HA 12CA5 (red) and anti-T444-P (green) antibodies. Nuclei are indicated in blue. D. COS-7 cells transfected with nucleus-targeted GFP (SV40NLS-GFP) were either left untreated (− OA) or incubated with okadaic acid (+ OA) before fixation. Nuclei are indicated in blue.

Membrane-targeted hMOB1A potently activates NDR1 at the plasma membrane. A. Lysates of COS-7 cells coexpressing the indicated combinations of HA-tagged wild-type (wt) or MOB1A-binding-deficient (Y31A) NDR1, myc-tagged hMOB1A, and membrane-targeted hMOB1A (mp-myc-hMOB1A) were analyzed by immunoprecipitation (IP) using anti-HA 12CA5 antibody. Complexes were assayed by Western blotting using anti-T444-P (top panel), anti-S281-P (middle top panel), and anti-HA antibody (middle bottom panel). Lysates were immunoblotted with anti-myc 9E10 antibody (bottom panel). B. In parallel, immunocomplexes were subjected to kinase assays. The robust increase of NDR1 activity by coexpression of membrane-targeted hMOB1A is marked in red. C. Cells transfected with the indicated NDR1 and hMOB1A constructs were subjected to S100/P100 fractionation (S, cytoplasm; P, membrane) before immunoblotting with anti-T444-P (top panel), anti-HA 12CA5 (middle panel), and anti-myc 9E10 (bottom panel) antibodies. D. The S100/P100 extracts described above for panel C were subjected to immunoprecipitation using anti-HA 12CA5 antibody before analysis by Western blotting using anti-T444-P (top panel), anti-S281-P (middle panel), and anti-HA (bottom panel) antibodies. An unspecific signal is indicated by an asterisk. E. In parallel, immunocomplexes were subjected to kinase assays. The increase of NDR1 activity in the membrane fraction is marked in red. F. Cells coexpressing HA-NDR1 wt/empty vector (top panels), HA-NDR1 wt/myc-hMOB1A (middle top panels), HA-NDR1 wt/mp-myc-hMOB1A (middle bottom panels), or HA-NDR1 (Y31A)/mp-myc-hMOB1A (bottom panels) were processed for immunofluorescence analysis with anti-T444-P (green), anti-myc 9E10 (red), and anti-HA 3F10 (blue) antibodies. Error bars indicate standard deviations.

Induced membrane association of hMOB1A results in rapid activation of NDR1. A. COS-7 cells expressing myc-tagged hMOB1A fused to the C1 domain of PKCα (myc-C1-hMOB1A) and wild-type (wt) HA-NDR1 or a MOB1A-binding deficient (Y31A) mutant were serum starved overnight before incubation with TPA for the indicated times. Cell lysates were subjected to S100/P100 fractionation (S, cytoplasm; P, membrane) before immunoblotting with anti-T444-P (top panel), anti-HA 12CA5 (middle panel), and anti-myc 9E10 (bottom panel) antibodies. B. Lysates of cells expressing HA-tagged wt or NDR1 (Y31A) and myc-C1-hMOB1A were analyzed by immunoprecipitation (IP) using anti-HA 12CA5 antibody. Complexes were assayed by Western blotting using anti-T444-P (top panel), anti-S281-P (top middle panel), and anti-HA (bottom middle panel) antibodies. Lysates were immunoblotted with anti-myc 9E10 antibody (bottom panel). Prior to lysis cells were incubated for 20 min with (+) or without TPA. C. In parallel, immunocomplexes were subjected to kinase assays. The significant elevation of NDR1 activity by stimulated membrane association of myc-C1-hMOB1A is highlighted in dark gray. Error bars indicate standard deviations.

Human NDR1 is a mainly cytoplasmic kinase. A. U2-OS cells expressing empty vector (lane 1), untagged human NDR1 (lane 2), or untagged human NDR2 (lane 3) were processed for immunoblotting using anti-NDR NT (top panel), anti-NDR1 (middle panel), and anti-α-tubulin (bottom panel) antibodies. Arrowheads indicate the positions of NDR1 and NDR2. B. U2-OS cells were incubated in the absence (lane 1) or presence (lane 2) of OA before processing for Western blotting using anti-Thr444-P (top panel), anti-NDR1 (middle panel), and anti-α-tubulin (bottom panel) antibodies. C. U2-OS cells were subjected to biochemical cell fractionation (T, total; N, nuclear; C, cytoplasmic; M, membrane) and processed for immunoblotting with anti-NDR1 (top panel), anti-lamin A/C (nuclear marker; top middle panel), anti-α-tubulin (cytoplasmic marker; bottom middle panel), and anti-p63/Climp (perinuclear/membrane marker; bottom panel) antibodies. D. COS-7 cells expressing HA-tagged NDR1 were subjected to biochemical cell fractionation (T, total; N, nuclear; C, cytoplasmic; M, membrane) and processed for immunoblotting with anti-HA (top panel), anti-lamin A/C (top middle panel), anti-α-tubulin (bottom middle panel), and anti-p63/Climp (bottom panel) antibodies. E. COS-7 and U2-OS cells expressing either GFP-NDR1 (top panels), HA-NDR1 (middle panels), or myc-NDR1 (bottom panels) were analyzed by indirect immunofluorescence microscopy using anti-HA Y11 (middle panels), anti-myc (bottom panels), or no (top panels) antibody. GFP/FITC signals are shown in green. Nuclei are in blue. F. COS-7 cells expressing HA-NDR1 were fixed with either methanol (MeOH) (top panels) or paraformaldehyde (PFA) (bottom panels) before permeabilization and antibody staining using anti-HA Y11 antibody. Arrowheads indicate NDR1 species found in plasma membrane structures. Enlargements of the regions indicated by white rectangles are shown on the right (Zoom).

Membrane-targeted NDR1 is partially active and can be further activated by coexpression of hMOB1A without OA treatment. A. COS-7 cells transfected with membrane-targeted NDR1 (mp-HA-NDR1) were either left untreated (− OA) or incubated with okadaic acid (+ OA), before processing for immunofluorescence using anti-HA Y11 (left panels). Nuclei are in blue. B. COS-7 cells were transfected with the indicated NDR1 plasmids and subjected to S100/P100 fractionation (S, cytoplasm; P, membrane) before immunoblotting with anti-T444-P (top panel) and anti-HA 12CA5 antibody (bottom panel). C. The S100/P100 extracts described above for panel B were subjected to immunoprecipitation using anti-HA 12CA5 antibody and subsequently to kinase assays. Elevated kinase activity was found only in mp-NDR1 membrane extracts (red bar). D. Lysates of COS-7 cells expressing HA-tagged wild-type (wt), membrane-tagged wt kinase-dead (kd), or hMOB1A-binding-deficient (Y31A) NDR1 were subjected to immunoprecipitation (IP) using anti-HA 12CA5 antibody. Complexes were analyzed by Western blotting using anti-T444-P (top panel), anti-S281-P (top middle panel), and anti-HA antibody (bottom middle panel). Prior to lysis, the indicated cells were incubated with OA (+). In certain samples, myc-tagged hMOB1A was coexpressed as verified by immunoblotting using anti-myc antibody (bottom panel). An unspecific signal is indicated by an asterisk. E. In parallel, immunocomplexes were subjected to kinase assays. Membrane-targeted NDR1 activity was about fivefold elevated compared to wild type and was further increased by hMOB1A coexpression (red bars). Error bars indicate standard deviations.

Determination of NDR1 localization domains. A. COS-7 cells expressing HA-tagged full-length (amino acids 1 to 465; top panels), N-terminally truncated (amino acids 83 to 465; middle panels), or C-terminally truncated (amino acids 1 to 380; bottom panels) NDR1 were processed for immunofluorescence using anti-HA Y11 antibody (green). Corresponding DNA stainings are shown on the right. The percentages of transfected cells displaying predominantly cytoplasmic signals are indicated. B. Cells transfected with plasmids encoding GFP-GFP alone (top panels), GFP-SV40NLS-GFP (middle panels), or GFP-NDR1(amino acids 262 to 278)-GFP (bottom panels) were subsequently analyzed by immunofluorescence. The percentages of stained cells as illustrated are indicated (>200 cells per construct were analyzed). Nuclei are shown in blue. The postulated NLS of NDR1 did not drive GFP into the nucleus, in contrast to the SV40 NLS. C. Cells expressing HA-tagged wild-type (wt), N-terminally truncated (amino acids 83 to 465), or C-terminally truncated (amino acids 1 to 380) NDR1, or selected mutants carrying point mutations at Leu92/Val94 (L92/V94A) or Leu160/Leu162 (L160/162A), were left untreated or incubated for 8 h with 10 ng/ml LMB or ethanol (EtOH), before processing for immunofluorescence using anti-HA Y11 antibody (green). The percentages of stained cells as shown are indicated (>200 cells per construct were analyzed). Nuclei are shown (right panels) and highlighted by white dashed lines in selected pictures. pNES, potential NES. D. COS-7 cells were transfected with the indicated HA-tagged NDR1 constructs and subjected to S100/P100 fractionation (T, total; S, cytoplasm; P, membrane), before immunoblotting with anti-HA 12CA5 antibody. E. Schematic representation of human NDR1 and mutant derivatives. The kinase domain (green) and the S100B/MOB association (SMA) (orange) domains are indicated. The N-terminal domain required for membrane association is marked by a red rectangle.