MyosinV controls PTEN function and neuronal cell size - PubMed (original) (raw)

. 2009 Oct;11(10):1191-6.

doi: 10.1038/ncb1961. Epub 2009 Sep 20.

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MyosinV controls PTEN function and neuronal cell size

Michiel T van Diepen et al. Nat Cell Biol. 2009 Oct.

Erratum in

Abstract

The tumour suppressor PTEN can inhibit cell proliferation and migration as well as control cell growth, in different cell types. PTEN functions predominately as a lipid phosphatase, converting PtdIns(3,4,5)P(3) to PtdIns(4,5)P(2), thereby antagonizing PI(3)K (phosphoinositide 3-kinase) and its established downstream effector pathways. However, much is unclear concerning the mechanisms that regulate PTEN movement to the cell membrane, which is necessary for its activity towards PtdIns(3,4,5)P(3) (Refs 3, 4, 5). Here we show a requirement for functional motor proteins in the control of PI3K signalling, involving a previously unknown association between PTEN and myosinV. FRET (Förster resonance energy transfer) measurements revealed that PTEN interacts directly with myosinV, which is dependent on PTEN phosphorylation mediated by CK2 and/or GSK3. Inactivation of myosinV-transport function in neurons increased cell size, which, in line with known attributes of PTEN-loss, required PI(3)K and mTor. Our data demonstrate a myosin-based transport mechanism that regulates PTEN function, providing new insights into the signalling networks regulating cell growth.

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Figures

Figure 1

Figure 1. Identification and characterisation of the PTEN:MyosinVa interaction

a, PTEN was immunoprecipitated (IP) from E18 rat brain and liver homogenate. Coomassie-stained gel of PTEN IP shows a brain-specific band of approximately 205 kD (star), which was identified as MyosinVa by tandem mass spectrometry. b, E18 rat brain extract was incubated with anti-PTEN antibody or control IgG; IPs were analysed with indicated antibodies. c, Domain structure of PTEN and PTEN-deletion constructs. d, HEK293 cells were co-transfected with GFP-PTEN (or indicated GFP-PTEN deletion constructs) and flag-MyosinVa globular domain (MVag). Following anti-flag immunoprecipitation, co-precipitates were analysed with anti-GFP (for PTEN) and anti-flag antibodies (for MVag). The empty flag vector (−) was used in control experiments. The PTEN:MyosinVa interaction is independent of the PTEN PDZ binding site (aa 400-403). The PTEN C2 domain alone does not support interaction (aa 182-353), whilst the PTEN C2 domain plus the C-terminal tail (aa 182-403) shows interaction with MVag. e, The PTEN:MyosinV interaction is regulated by phosphorylation. Left; alanine substitutions of S380, T382, T383 (PTEN AAA) reduces the PTEN:MyosinVa interaction in comparison to substitution of the amino acids to aspartic acids (PTEN DDD). Right; HEK293 cells co-expressing GFP-PTEN and MVag were treated with the CK2 inhibitor 5,6-Dichlorobenzimidazole Riboside (DRB; 10 μM) or the GSK3 inhibitor CT99021 (2 μM), before co-immunoprecipitation as described in d. f, Analysis of the PTEN:MyosinVa interaction by FRET in PC12 cells. Images show the lifetime maps of spatial FRET across cells using a pseudocolour scale (blue, normal GFP lifetime; red, FRET). GFP-PTEN alone (top left) demonstrates normal GFP lifetime in the absence of acceptor, all other images show co-expression of GFP-PTEN and mCherry-MVag. In the lower panels, cells were treated with DRB (left), or DRB and CT99021 (right). g, Bar graph representing the average FRET efficiency of 12 cells (4 cells in 3 independent experiments) ± sem. *p<0.02; **p<0.002.

Figure 2

Figure 2. MyosinV regulates neuronal soma size through PI3K signalling

Hippocampal neurons from E15.5 wild type (wt) or dilute lethal (dilute) mice were cultured for 7 days in vitro (DIV) and transfected with different constructs before fixation at 14 DIV. Where indicated, LY294002 (10 μM) or Rapamycin (200 nM) was applied 2 hours after transfection. a, Confocal images of neurons labelled with anti-Map-2 antibody (red); GFP (green) visualizes Ires-GFP or MVag-IresGFP expression. b, c, Quantification of neuronal soma size. For measurements, images were taken on an epifluorescence microscope, and examined using SimplePCI software after manually delineating the cell margins. Each data point represents the relative neuronal soma area compared to wt IresGFP expressing neurons ± sem, which was set to 1. n=4 independent experiments (n=6 for wt and dilute neurons, Ires-GFP or MVag-IresGFP), in each experiment at least 30 neurons were assessed. *p<0.001 (Student’s T-test). d, Expression of MVag in cortical neurons in vivo increases neuronal soma size. Coronal sections of E15.5 mouse cortices electroporated with control GFP (left) or GFP/MVag (right) at E13.5. e, Quantification of neuronal soma size in vivo. Only neurons undergoing radial migration were evaluated. n=3 independent control experiments and 4 MVag experiments, in each experiment at least 70 neurons were assessed. *p<0.001

Figure 3

Figure 3

The PTEN:MyosinV interaction competes with PTEN’s closed conformation, and is mediated by a positively charged region located in the globular domain of MyosinV. a, Domain structure of PTEN and PTEN-deletion constructs used in co-immunoprecipitation experiments. C-terminal PTEN phosphorylation sites (P-sites) involved in mediating intramolecular PTEN and MyosinV binding are indicated. b, the PTEN:MyosinVa interaction can be competed for by the PTEN N-terminal region. HEK293 cells were co-transfected with flag-MyosinVa globular domain (MVag) and the GFP-PTEN C2+tail mutant, in the presence of increasing amounts of GFP-PTEN N. Following anti-flag immunoprecipitation, precipitates were analysed with anti-flag antibodies (for MVag) and with an anti-PTEN antibody recognising the PTEN C-terminus, only. c, Alignment of positively charged amino acid clusters present in the globular domains of MyosinVa, MyosinVb and MyosinVc. MVa* and MVa** denotes MVa RKR↦QNI and MVa KKK↦QNI mutants, respectively. d, Essential requirement of the KKK cluster for the PTEN:MyosinV interaction. Flag-MVag, flag-MVag* or flag-MVag** were co-expressed with GFP-PTEN as before and following anti-flag immunoprecipitation, precipitates were analysed with anti-flag antibodies (for MVags). The empty flag vector (−) was used in control experiments. e, Requirement of the KKK cluster for MVag induced increases in cell size (Fig. 2). Hippocampal neurons were cultured and transfected with control IresGFP, MVag, MVag*, or MVag** as before. Each data point represents the relative neuronal soma area compared to control (IresGFP) expressing neurons ± sem, n=3 independent experiments, in each experiment at least 40 neurons were assessed. *p<0.0001. f, Hippocampal neurons from E15.5 mice were cultured for 7 days in vitro (DIV) and transfected with MVag-IresGFP before fixation at 14 DIV. Phase contrast image was overlaid with GFP to visualise neuronal soma increase in MVag expressing, in comparison to non-expressing, neurons in the same culture.

Figure 4

Figure 4. Neuronal soma size is controlled by GSK3 and CK2 upstream of PTEN and MyosinV

Hippocampal neurons were cultured and transfected as described in Figure 2. Control IresGFP (#) was expressed in all indicated treatments. CT99021 (2 μM) or DRB (10 μM) was applied 2 hours after transfection. a, Quantification of neuronal soma size at 14 DIV. Each data point represents the relative neuronal soma area compared to control (IresGFP) expressing neurons ± sem, n=3 independent experiments, in each experiment ≥ 40 neurons were assessed. *p<0.03, **p<0.0001. b, Schematic illustrating the signalling relationship. c, Neuronal cultures were labelled with anti-PS6 antibody (left; shown in red in the merge); green neurons in the merged image (arrowhead) visualize neurons expressing indicated constructs.

Comment in

References

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