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The Caenorhabditis elegans Eph receptor activates NCK and N-WASP, and inhibits Ena/VASP to regulate growth cone dynamics during axon guidance.

Mohamed AM, Boudreau JR, Yu FP, Liu J, Chin-Sang ID - PLoS Genet. (2012)

Bottom Line: We identified NCK-1 and WSP-1/N-WASP as downstream effectors of VAB-1.Furthermore, VAB-1, NCK-1, and WSP-1 can form a complex in vitro.We suggest that VAB-1/Eph RTK can stop axonal outgrowth by inhibiting filopodia formation at the growth cone by activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex and by inhibiting UNC-34/Ena activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Queen's University, Kingston, Canada.

ABSTRACT
The Eph receptor tyrosine kinases (RTKs) are regulators of cell migration and axon guidance. However, our understanding of the molecular mechanisms by which Eph RTKs regulate these processes is still incomplete. To understand how Eph receptors regulate axon guidance in Caenorhabditis elegans, we screened for suppressors of axon guidance defects caused by a hyperactive VAB-1/Eph RTK. We identified NCK-1 and WSP-1/N-WASP as downstream effectors of VAB-1. Furthermore, VAB-1, NCK-1, and WSP-1 can form a complex in vitro. We also report that NCK-1 can physically bind UNC-34/Enabled (Ena), and suggest that VAB-1 inhibits the NCK-1/UNC-34 complex and negatively regulates UNC-34. Our results provide a model of the molecular events that allow the VAB-1 RTK to regulate actin dynamics for axon guidance. We suggest that VAB-1/Eph RTK can stop axonal outgrowth by inhibiting filopodia formation at the growth cone by activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex and by inhibiting UNC-34/Ena activity.

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Related in: MedlinePlus

NCK-1 physically interacts with VAB-1 and UNC-34.(A) The NCK-1 SH2 domain binds to the Y673 of VAB-1. Yeast-two hybrid assays shows that the NCK-1 SH2 domain (Prey) can bind wild-type (WT) VAB-1 (669 aa–985 aa), but fails to interact when VAB-1 Y673 is changed to glutamic acid (Y673E). (B–C) GST pull-down assays. L = Load; U = unbound fraction; B = bound fraction. (B) Different NCK-1 domains fused to GST show that the SH2 domain is required for VAB-1 binding. The three SH3 domains alone do not bind VAB-1. (C) Bacterial expressed MBP-VAB-1 (intracellular region) has tyrosine kinase activity and shows autophosphorylation. The point mutation G912E in the VAB-1 kinase domain abolishes the VAB-1 kinase activity. The asterisks indicate break down products during purification. Phosphotyrosine was detected using anti-phosphotyrosine antibodies (4G10). The blot was stained with Ponceau S to show equal protein loading (below). (D) The binding of SH2 domain of NCK-1 to VAB-1 is kinase dependent. The kinase inactive VAB-1(G912E) failed to interact with the SH2 domain of NCK-1. (E) The NCK-1 SH2 domain shows high specificity for VAB-1. Yeast-two hybrid assays of other SH2 domains failed to interact with VAB-1. (F–G) GST-NCK-1 pull-down assays with MBP-UNC-34. (F) UNC-34 binds to NCK-1, both the proline rich (PRO) and EVH2 domains are required to bind NCK-1 (not shown). (G) Different GST-NCK-1 domains show that all SH3 domains can independently interact with full length UNC-34. The asterisks marks the correct protein fragment expected, and all other fragments below the marked are break down products. The dashed lines in E and F indicate a cropped region from the same blot.
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pgen-1002513-g004: NCK-1 physically interacts with VAB-1 and UNC-34.(A) The NCK-1 SH2 domain binds to the Y673 of VAB-1. Yeast-two hybrid assays shows that the NCK-1 SH2 domain (Prey) can bind wild-type (WT) VAB-1 (669 aa–985 aa), but fails to interact when VAB-1 Y673 is changed to glutamic acid (Y673E). (B–C) GST pull-down assays. L = Load; U = unbound fraction; B = bound fraction. (B) Different NCK-1 domains fused to GST show that the SH2 domain is required for VAB-1 binding. The three SH3 domains alone do not bind VAB-1. (C) Bacterial expressed MBP-VAB-1 (intracellular region) has tyrosine kinase activity and shows autophosphorylation. The point mutation G912E in the VAB-1 kinase domain abolishes the VAB-1 kinase activity. The asterisks indicate break down products during purification. Phosphotyrosine was detected using anti-phosphotyrosine antibodies (4G10). The blot was stained with Ponceau S to show equal protein loading (below). (D) The binding of SH2 domain of NCK-1 to VAB-1 is kinase dependent. The kinase inactive VAB-1(G912E) failed to interact with the SH2 domain of NCK-1. (E) The NCK-1 SH2 domain shows high specificity for VAB-1. Yeast-two hybrid assays of other SH2 domains failed to interact with VAB-1. (F–G) GST-NCK-1 pull-down assays with MBP-UNC-34. (F) UNC-34 binds to NCK-1, both the proline rich (PRO) and EVH2 domains are required to bind NCK-1 (not shown). (G) Different GST-NCK-1 domains show that all SH3 domains can independently interact with full length UNC-34. The asterisks marks the correct protein fragment expected, and all other fragments below the marked are break down products. The dashed lines in E and F indicate a cropped region from the same blot.

Mentions: In a parallel approach we used yeast two-hybrid screens to identify effectors of VAB-1/Eph RTK signaling and identified the full length NCK-1 as a binding partner of the VAB-1 intracellular kinase region. Yeast two-hybrid analysis showed that the NCK-1 SH2 domain is sufficient to bind VAB-1 and that VAB-1 tyrosine Y673 is crucial for the interaction with the NCK-1 SH2 domain (Figure 4A).


The Caenorhabditis elegans Eph receptor activates NCK and N-WASP, and inhibits Ena/VASP to regulate growth cone dynamics during axon guidance.

Mohamed AM, Boudreau JR, Yu FP, Liu J, Chin-Sang ID - PLoS Genet. (2012)

NCK-1 physically interacts with VAB-1 and UNC-34.(A) The NCK-1 SH2 domain binds to the Y673 of VAB-1. Yeast-two hybrid assays shows that the NCK-1 SH2 domain (Prey) can bind wild-type (WT) VAB-1 (669 aa–985 aa), but fails to interact when VAB-1 Y673 is changed to glutamic acid (Y673E). (B–C) GST pull-down assays. L = Load; U = unbound fraction; B = bound fraction. (B) Different NCK-1 domains fused to GST show that the SH2 domain is required for VAB-1 binding. The three SH3 domains alone do not bind VAB-1. (C) Bacterial expressed MBP-VAB-1 (intracellular region) has tyrosine kinase activity and shows autophosphorylation. The point mutation G912E in the VAB-1 kinase domain abolishes the VAB-1 kinase activity. The asterisks indicate break down products during purification. Phosphotyrosine was detected using anti-phosphotyrosine antibodies (4G10). The blot was stained with Ponceau S to show equal protein loading (below). (D) The binding of SH2 domain of NCK-1 to VAB-1 is kinase dependent. The kinase inactive VAB-1(G912E) failed to interact with the SH2 domain of NCK-1. (E) The NCK-1 SH2 domain shows high specificity for VAB-1. Yeast-two hybrid assays of other SH2 domains failed to interact with VAB-1. (F–G) GST-NCK-1 pull-down assays with MBP-UNC-34. (F) UNC-34 binds to NCK-1, both the proline rich (PRO) and EVH2 domains are required to bind NCK-1 (not shown). (G) Different GST-NCK-1 domains show that all SH3 domains can independently interact with full length UNC-34. The asterisks marks the correct protein fragment expected, and all other fragments below the marked are break down products. The dashed lines in E and F indicate a cropped region from the same blot.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3285579&req=5

pgen-1002513-g004: NCK-1 physically interacts with VAB-1 and UNC-34.(A) The NCK-1 SH2 domain binds to the Y673 of VAB-1. Yeast-two hybrid assays shows that the NCK-1 SH2 domain (Prey) can bind wild-type (WT) VAB-1 (669 aa–985 aa), but fails to interact when VAB-1 Y673 is changed to glutamic acid (Y673E). (B–C) GST pull-down assays. L = Load; U = unbound fraction; B = bound fraction. (B) Different NCK-1 domains fused to GST show that the SH2 domain is required for VAB-1 binding. The three SH3 domains alone do not bind VAB-1. (C) Bacterial expressed MBP-VAB-1 (intracellular region) has tyrosine kinase activity and shows autophosphorylation. The point mutation G912E in the VAB-1 kinase domain abolishes the VAB-1 kinase activity. The asterisks indicate break down products during purification. Phosphotyrosine was detected using anti-phosphotyrosine antibodies (4G10). The blot was stained with Ponceau S to show equal protein loading (below). (D) The binding of SH2 domain of NCK-1 to VAB-1 is kinase dependent. The kinase inactive VAB-1(G912E) failed to interact with the SH2 domain of NCK-1. (E) The NCK-1 SH2 domain shows high specificity for VAB-1. Yeast-two hybrid assays of other SH2 domains failed to interact with VAB-1. (F–G) GST-NCK-1 pull-down assays with MBP-UNC-34. (F) UNC-34 binds to NCK-1, both the proline rich (PRO) and EVH2 domains are required to bind NCK-1 (not shown). (G) Different GST-NCK-1 domains show that all SH3 domains can independently interact with full length UNC-34. The asterisks marks the correct protein fragment expected, and all other fragments below the marked are break down products. The dashed lines in E and F indicate a cropped region from the same blot.
Mentions: In a parallel approach we used yeast two-hybrid screens to identify effectors of VAB-1/Eph RTK signaling and identified the full length NCK-1 as a binding partner of the VAB-1 intracellular kinase region. Yeast two-hybrid analysis showed that the NCK-1 SH2 domain is sufficient to bind VAB-1 and that VAB-1 tyrosine Y673 is crucial for the interaction with the NCK-1 SH2 domain (Figure 4A).

Bottom Line: We identified NCK-1 and WSP-1/N-WASP as downstream effectors of VAB-1.Furthermore, VAB-1, NCK-1, and WSP-1 can form a complex in vitro.We suggest that VAB-1/Eph RTK can stop axonal outgrowth by inhibiting filopodia formation at the growth cone by activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex and by inhibiting UNC-34/Ena activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Queen's University, Kingston, Canada.

ABSTRACT
The Eph receptor tyrosine kinases (RTKs) are regulators of cell migration and axon guidance. However, our understanding of the molecular mechanisms by which Eph RTKs regulate these processes is still incomplete. To understand how Eph receptors regulate axon guidance in Caenorhabditis elegans, we screened for suppressors of axon guidance defects caused by a hyperactive VAB-1/Eph RTK. We identified NCK-1 and WSP-1/N-WASP as downstream effectors of VAB-1. Furthermore, VAB-1, NCK-1, and WSP-1 can form a complex in vitro. We also report that NCK-1 can physically bind UNC-34/Enabled (Ena), and suggest that VAB-1 inhibits the NCK-1/UNC-34 complex and negatively regulates UNC-34. Our results provide a model of the molecular events that allow the VAB-1 RTK to regulate actin dynamics for axon guidance. We suggest that VAB-1/Eph RTK can stop axonal outgrowth by inhibiting filopodia formation at the growth cone by activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex and by inhibiting UNC-34/Ena activity.

Show MeSH
Related in: MedlinePlus