|MyosinV controls PTEN function and neuronal cell size.|
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|PMID: 19767745 Owner: NLM Status: MEDLINE|
|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.|
|Michiel T van Diepen; Maddy Parsons; C Peter Downes; Nicholas R Leslie; Robert Hindges; Britta J Eickholt|
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|Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2009-09-20|
|Title: Nature cell biology Volume: 11 ISSN: 1476-4679 ISO Abbreviation: Nat. Cell Biol. Publication Date: 2009 Oct|
|Created Date: 2009-10-01 Completed Date: 2009-11-17 Revised Date: 2010-09-27|
Medline Journal Info:
|Nlm Unique ID: 100890575 Medline TA: Nat Cell Biol Country: England|
|Languages: eng Pagination: 1191-6 Citation Subset: IM|
|MRC Centre for Developmental Neurobiology, New Hunt's House, King's College London, London SE1 1UL, UK.|
|APA/MLA Format Download EndNote Download BibTex|
Alanine / metabolism
Amino Acid Sequence
Amino Acid Substitution
Aspartic Acid / metabolism
Binding Sites / genetics
Glycogen Synthase Kinase 3 / metabolism
Green Fluorescent Proteins / metabolism
Hippocampus / cytology
Molecular Sequence Data
Myosin Type V / chemistry, metabolism*
Neurons / cytology*, metabolism
PTEN Phosphohydrolase / genetics, metabolism*
Phosphoproteins / metabolism
Protein Binding / genetics, physiology
Protein Structure, Tertiary
Sequence Homology, Amino Acid
Signal Transduction / genetics, physiology
|BB/C514307/1//Biotechnology and Biological Sciences Research Council; BB/C514307/1//Biotechnology and Biological Sciences Research Council; G9403619//Medical Research Council|
|0/CKT2 protein, mouse; 0/Phosphoproteins; 147336-22-9/Green Fluorescent Proteins; 56-41-7/Alanine; 56-84-8/Aspartic Acid; EC 22.214.171.124/1-Phosphatidylinositol 3-Kinase; EC 126.96.36.199/Glycogen Synthase Kinase 3; EC 188.8.131.52/Pten protein, mouse; EC 184.108.40.206/PTEN Phosphohydrolase; EC 3.6.1.-/Myosin Type V|
|Nat Cell Biol. 2009 Oct;11(10):1177-9
|Nat Cell Biol. 2009 Nov;11(11):1387
Journal ID (nlm-journal-id): 100890575
Journal ID (pubmed-jr-id): 21417
Journal ID (nlm-ta): Nat Cell Biol
Journal ID (iso-abbrev): Nat. Cell Biol.
nihms-submitted publication date: Day: 4 Month: 8 Year: 2009
Electronic publication date: Day: 20 Month: 9 Year: 2009
Print publication date: Month: 10 Year: 2009
pmc-release publication date: Day: 01 Month: 4 Year: 2010
Volume: 11 Issue: 10
First Page: 1191 Last Page: 1196
PubMed Id: 19767745
|MyosinV controls PTEN function and neuronal cell size|
|Michiel van Diepen1|
|C. Peter Downes3|
|Nicholas R. Leslie3|
|Britta J Eickholt1|
1MRC Centre for Developmental Neurobiology, New Hunt’s House, King’s College London, London SE1 1UL, United Kingdom
2The Randall Division of Cell and Molecular Biophysics, New Hunt’s House, King’s College London, London SE1 1UL, United Kingdom
3Division of Molecular Physiology, College of Life Sciences, University of Dundee, Dundee, Scotland, DD1 5EH, UK
|Correspondence: Correspondence should be addressed to BJE. Britta.J.Eickholt@kcl.ac.uk; requests for material should be addressed to BJE or NRL (email@example.com)
Author contributions All authors designed experiments. MvD, MP, RH and BJE performed research. BJE wrote the manuscript.
Although sub-cellular distribution of PTEN has been reported to regulate important aspects of PI3K signalling in Dictyostelium, epithelial cells, neutrophils and neurons8-11, the determining cellular machinery remains to be established. We set out to identify proteins involved in regulating the sub-cellular localisation and/or function of PTEN in neurons. Following PTEN immunoprecipitation from brain and liver, PAGE revealed a prominent brain-specific band of approximately 205 kD that was identified as MyosinVa by mass spectrometry (Fig. 1a). The PTEN:MyosinVa association was confirmed using co-immunoprecipitation from brain lysates (Figure 1b). MyosinVa is a plus-end directed, processive motor, which transports mRNA, proteins and multiple organelles along actin filaments into the cell periphery, predominantly through interactions with its globular, cargo-binding domain12. We co-expressed flag-tagged MyosinVa globular domain (MVag) with GFP-PTEN in HEK293 cells and co-immunoprecipitated PTEN with MyosinVa (Fig. 1d). To identify the region within PTEN that mediates the association, flag-MVag was co-expressed with different GFP-tagged PTEN deletion mutants (Fig. 1c, d). The results indicated that the interaction required PTEN aa354-399, which contains a cluster of phosphorylation sites regulated by CK2 and GSK3 (Fig. 1c, d)13, 14. To test if the interaction depends on C-terminal PTEN phosphorylation we used mutant PTEN constructs in which Ser380, Thr382, and Thr383 are mutated to alanines (PTEN AAA) or aspartic acid (PTEN DDD) and found that PTEN:MyosinVa appears to be greatly reduced in PTEN AAA, whilst PTEN DDD was as efficient in associating with MyosinVa as wild type PTEN (Fig, 1e). We then applied inhibitors specific for CK2 (DRB) and/or GSK3 (CT99021) to GFP-PTEN/flag-MVag expressing cells and performed immunoprecipitations as before. The PTEN:MyosinVa association was sensitive to individual inhibition of CK2 or GSK3, and reduced further when both kinase inhibitors were present (Fig. 1e). Thus, the PTEN:MyosinVa interaction depends on the phosphorylation of residues at the PTEN C-terminus, a mechanism previously demonstrated to regulate binding of PTEN with neutral endopeptidase15.
To analyse localisation and regulation of the protein:protein interaction in intact cells we measured fluorescence resonance energy transfer (FRET) between PTEN and MyosinVa. We expressed GFP-PTEN and mCherry-MVag in PC12 cells and analysed FRET by fluorescence lifetime imaging microscopy (FLIM)16. Data demonstrated reproducible spots in GFP lifetime reduction, indicative of direct PTEN:MyosinVa interaction that were confined to small patches in the cell periphery (Fig. 1f). In agreement with our co-immunoprecipitation experiments we found that treatment with DRB and/or CT99021 reduced PTEN:MyosinVa FRET efficiencies (Fig. 1f, g). Thus, PTEN interacts directly with MyosinVa in a spatially regulated manner, dependent on the activity of CK2 and GSK3.
MyosinVa is highly enriched in the nervous system (Supplementary Fig. 1), where it localizes within neuronal growth cones and dendritic spines17-19. In humans, mutations in MyosinVa cause Griscelli syndrome type 1 and Elejalde syndrome, which are both characterized by hypopigmentation of the skin and hair, as well as prepubescent onset of severe neurological abnormalities, including seizures and mental retardation20. Similarly, mice lacking functional MyosinVa due to homozygous deletion of dilute lethal Myo5ad-1 - hereafter referred to as dilute lethal mice - show neurological abnormalities17. We utilized dilute lethal mice to test the possibility that MyosinVa mediates PTEN movement toward the membrane. In this case MyosinVa deficiencies would phenocopy loss of PTEN, potentially generating neurons in the cerebral cortex and hippocampus with enlarged soma, ectopic processes and increased numbers of synapses6, 7, 21, 22. However, gross morphology of neurons in the cortex and the hippocampus at postnatal day (P) 16 was normal in dilute lethal mice (Supplementary Fig. 2). In addition to MyosinVa, two other class members, MyosinVb and MyosinVc, have been identified23, 24 and co-immunoprecipitation experiments reveals interactions with PTEN (Supplementary Fig. 3). MyosinVb, but not MyosinVc, is expressed in the developing brain (Supplementary Fig. 4), raising the possibility for functional compensation following loss of MyosinVa. Therefore, we exploited the MyosinVa globular domain, which is known to block MyosinVa-mediated transport25, and, when present in excess, should compete for PTEN interactions to other MyosinVs. IresGFP or MVag-IresGFP were expressed in hippocampal neurons cultured from wt mice and dilute lethal littermates at 7 days in vitro (DIV) and neuronal morphology was assessed at 14 DIV. MVag-IresGFP induced a significant increase in neuronal soma size (Fig. 2a, b), which was antagonised by the PI3K inhibitor LY294002 (Fig. 2a, b). This result provides functional evidence that MyosinV regulates PI3K signalling, possibly by coordinating membrane transport of PTEN. To test this idea, we forced PTEN to the membrane by myristoylation (myrPTEN), which uncouples MyosinV-mediated transport from PTEN function. Expression of myrPTEN inhibited MVag’s effect on soma size (Fig. 2c). We then tested the effect of the mTor inhibitor rapamycin, an established downstream signalling component of neuronal hypertrophy in PTEN deficient brains6. Rapamycin treatment reduced soma size in IresGFP and also in MVag expressing neurons (Fig. 2c). We also complemented the dominant negative MVag approach by silencing MyosinVb in neurons. We co-transfected hippocampal neurons cultured from wt mice and dilute lethal littermates with IresGFP in the presence of unspecific control siRNA or MyosinVb specific siRNA, which resulted in a small but significant increase in soma sizes in neurons obtained from dilute lethal mice, only (Supplementary Fig. 5). These data demonstrate that loss of MyosinV transport function through overexpression of MVag – as well as through functional inactivation of the two main MyosinVs in hippocampal neurons - mimic PTEN deficiencies with respect to soma size. Collectively, these results are consistent with a model in which MyosinV-transport regulates PI3K signalling through interaction with PTEN.
To study if MyosinV controls neuronal cell size in vivo, we injected the constructs expressing MVag and GFP, or GFP alone, into the lateral ventricle of E13.5 mouse embryos and performed in utero electroporation26. After normal embryonic development in vivo for two days, many GFP expressing neurons adopted morphologies typical for their movement along the radial fibre scaffold, with a leading process extending toward the cortical plate and an axon trailing toward the ventricular zone (Fig. 2d). MVag expression permitted radial orientation in the majority of neurons, although the calibre of the leading process appeared to be enlarged (Fig. 2d). Most notably, MVag expressing neurons showed a significant increase in the size of their somas (Fig. 2d, e). Thus, functional MyosinVs are also essential for proper control of neuronal cell size in vivo.
C-terminal PTEN phosphorylation has previously been shown to induce a conformational switch of the protein, keeping the enzyme in a ‘closed’ formation with decreased membrane localisation and biological activity27, 28. Our results demonstrating that PTEN binds preferentially with MyosinVa in its phosphorylated state may indicate competitions between this intramolecular PTEN interaction and an interaction with MyosinV. In order to test this idea we examined if the association of the PTEN C2+tail mutant with flag-MVag (see Fig.1d) can be blocked by an N-terminal fragment of PTEN (PTEN N, aa 1-317) (Fig. 3a). We co-expressed MVag, the GFP-PTEN C2+tail mutant and increasing levels of PTEN N in HEK293 cells, and co-immunoprecipitated the PTEN tail mutant with MyosinVa. While PTEN N failed to interact with MVag (not shown here), increasing levels of this mutant competed for the PTEN tail association with the Myosin globular domain (Fig. 3b). Phosphorylation-dependent interactions of the PTEN C-terminal tail have been reported to require a cluster of three positively charged amino acids15, 27. We identified two similar clusters with high degree in conservation amongst all MyosinV class members (Fig. 3c), and mutated them individually to uncharged aa glutamine, asparagine, and isoleucine (MVa* RKR↦QNI; MVa** KKK↦QNI), as previously15. Only the second cluster (KKK) was essential for MVag’s association to PTEN in immunoprecipitation experiments, and – significantly – also for increases in neuronal soma size of native MVag-IresGFP (Fig. 3d-f). These experiments indicate that PTEN:MyosinV is based on electrostatic interactions between PTEN and a positively charged region located in the globular domain of MyosinV, which is in competition with PTEN’s closed conformation.
We next investigated potential upstream regulators of the PTEN:MyosinV interaction in the control of neuronal cell size. We showed that the PTEN:MyosinVa interaction is antagonized following inhibition of GSK3 and CK2 (Fig. 1), which indicates that these kinases may negatively regulate neuronal soma size upstream of PTEN transport. To test this hypothesis, we applied DRB and CT99021 to hippocampal neurons for 7 days, before analysis of soma size. Inhibition of CK2 and GSK3 led to soma size increases in IresGFP expressing neurons, but not in neurons expressing myrPTEN (Fig. 4a). In mice, neuron-specific overexpression of constitutive-active GSK3 decreases the brain volume, resulting in microcephaly due to reduced sizes of neurons29. Correspondingly, we find that somas of hippocampal neurons were significantly smaller following overexpression of the constitutive-active GSK3βS9A mutant (Fig. 4a). In contrast, perturbation of MyosinV-transport by co-expressing MVag with GSK3β S9A maintained MVag-enlarged neuronal soma size, which places GSK3 activity upstream of MyosinV transport and cell size regulation. To further substantiate this signalling relationship, we co-expressed a myristoylated phosphatase-dead PTEN mutant (myrPTEN C124S) with GSK3β S9A. Expression of myrPTEN C124S alone increased neuronal soma size (Fig. 4a), and we reasoned that in combination with GSK3β S9A, myrPTEN C124S signifies unequivocally whether GSK3 operates upstream or downstream of PTEN:MyosinV in controlling soma size. The myrPTEN C124S phenotype was dominant (Fig. 4a), which highlights a requirement for functional PTEN to decrease growth resulting from overactivation of GSK3.
We next assessed whether the presented evidence for GSK3 in controlling neuronal soma size by regulating the PTEN:MyosinV interaction can be correlated with the activity of the growth-control signalling machinery. Cell size enlargements caused by PTEN-deficiencies require mTor6, and correlate with elevated phosphorylation of its effector ribosomal protein S67. Hippocampal neurons maintain robust pS6 signals, which remain unaffected by expression of IresGFP (Fig. 4c). As expected, rapamycin treatment resulted in decreased pS6-labelling, confirming the specificity of this approach. Similarly, GSK3β S9A expression was accompanied by decreased pS6-labelling, whilst co-expression of GSK3β S9A with MVag antagonised GSK3-induced reduction in pS6 (Fig. 4c). Neurons expressing myrPTEN C124S, or myrPTEN C124S in combination with GSK3β S9A both exhibited marked amplification of pS6 in comparison to non-transfected neurons (Fig. 4c). Collectively, these results establish a causal link between GSK3 as upstream regulators of the PTEN:MyosinV interaction in the control of neuronal soma size and mTor signalling.
We have demonstrated a transport mechanism that controls PI3K signalling and neuronal soma size, which involves direct interaction of MyosinV with the tumour suppressor PTEN. This interaction is likely to permit a degree of cellular signalling integration, since PTEN associates with MyosinV – as well as antagonises PI3K signalling and functions – when GSK3 is active. Consistent with an established role for PI3K in negatively regulating GSK3 itself, our identification of a mechanism by which GSK3 phosphorylation directs PTEN function provides a possible novel positive feedback system that operates in concert with CK2 (Fig. 4b). Such organization offers a powerful way to amplify or antagonise PI3K signalling thresholds required for neurons to respond to extracellular signals involved in determining and maintaining proper neuronal soma size in the brain.
For in utero electroporation, timed pregnant E13.5 CD1 mice were used (the morning of the vaginal plug was E0.5). Experiments were performed following institutional guidelines and approved protocols. pCAβ-IRES-eGFPm5-grip was used as a control for pCAβ-MVag-IRES-eGFPm5-grip. One μl of purified DNA (1-2μg/μl) mixed with 0.005% Fast Green was injected in utero into the lateral ventricle and electroporated into the neocortical ventricular zone according to published protocols26. Brains were PFA-fixed and vibratome-sectioned for analyses by confocal microscopy.
In order to determine the spatial interaction between PTEN and MyosinVA, PC12 cells were transfected with p-mCherry-MVag and/or PTEN-EGFP-c2, differentiated for 2 days with NGF (50 ng/ml) and fixed for FLIM experiments to measure Förster resonance energy transfer FRET. Time-domain FLIM experiments and data analysis were performed as described previously16.
The globular domain from chick MyosinVa (MVag) was amplified by PCR (Proofstart;QIAGEN) (primers: 5′-GCCACCATGAGTCTGCAGCATGAGATCACC-3′ and 5′-GACACGTGATATGAAACCCAG-3′) and cloned into pCAβ-IRES-eGFPm5-grip, p-mCherry n1 (Clontech) or p3XFLAG-CMV™-7.1 (Sigma). Similarly, Mouse cDNA was used to clone the globular domain of MyosinVb (primers: 5′- ATGTCCACCATCAACGGCATTAAG-3′ and TTGACTTCATTGAGAAACTCCAG-3′) and MyosinVc (primers: 5′-ATGAGTGCTATTAACGGCATCAAGC-3′ and 5′- TTCAGTCTCCGGAGAAAGCCG-3′) into p3XFLAG-CMV™-7.1. Site directed mutagenesis of parental flag-MVag or MVag-IresGFP vectors were performed using the QuikChange II XL mutagenesis kit (Stratagene) according to the manufacturer’s protocols. The primers used were 5′-GGGTGAAACCAACAGGGCTGCAAAACATAACATCCAGCATTGCTGATGA-3′ and 5′-TCATCAGCAATGCTGGATGTTATGTTTTGCAGCCCTGTTGGTTTCACCC-3′ for the MVa RKR↦QNI mutation, and 5′-GCTGCACAGTTGTTGCAAGTGCAAAACATAACAGATGAAGATGCA-3′ and 5′-GCTTCTGCATCTTCATCTGTTATGTTTTGCACTTGCAACAACTGT-3′ for the MVa KKK↦QNI mutation. All mutations were validated by sequencing.
Cortices or livers from E18 rat embryos were homogenized in lysis buffer (50 mM Tris pH 7.5; 150 mM NaCl; 1% Triton X-100; 2 μM NaOV and 500 μM NaF) containing protease inhibitors (Roche). Immunoprecipitations were performed overnight (4°C) using 5μg of anti-PTEN antibody (A2B1, Santa Cruz) or mouse control antibody and protein-G beads (PIERCE). After washing, beads were boiled in loading buffer and separated on 10% SDS-polyacrylamide gels, which were fixed and stained using Brilliant Blue G - Colloidal Concentrate (Sigma) according to manufacturer’s instructions. Selected protein bands were isolated, processed and sequenced by Proteome Sciences plc. In short, after in gel trypsin digestion, samples were analysed by LC/MS/MS, the mass spectral data was processed into peak lists and searched against the Swiss Prot and NCBI non-redundant databases using Mascot software (Matrix Science, UK). For co-immunoprecipitation experiments, HEK293 cells were transfected with different constructs using Lipofectamine 2000 and anti-FLAG® M2 Affinity Gel (Sigma) was used for isolating the 3xFlag-MVag complex. Western blot analysis of immunoprecipitates was performed using anti-flag (Sigma), anti-GFP (Abcam) or anti-PTEN antibodies (Santa Cruz or Cell Signaling).
Cortices from E15.5 dilute lethal and wildtype littermates were used to isolate total RNA (Qiagen RNeasy mini kit), which was reversed transcribed into random primed cDNA (Reverse Transcription Kit, Promega) and served as template for all qPCR experiments. MyosinVa, MyosinVb, MyosinVc and PTEN mRNA levels were measured relative to Gapdh, Pgk1 and Hprt. Primers for qPCR analysis of the different mRNAs were ordered from QIAGEN (QuantiTect Primer Assays), Quantifast SYBR Green (QIAGEN) was used as a reporter for detection with the Light Cycler1.0 (Roche). All reactions were performed according to the manufacturer’s instructions.
FN2Author information The authors declare no competing financial interest.
This work was funded by the Biotechnology and Biological Science Research Council to BJE (BB/C514307/1), an MRC program grant to CPD (G9403619). MP is a Royal Society University Research Fellow. We thank John Hammer 3rd for providing the MyosinVa cDNA, Calum Sutherland for the GSK3 inhibitor CT99021, Eric Blanc for help with statistical analyses and Frank Gertler for comments on the manuscripts.
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