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Extracellular matrix-specific Caveolin-1 phosphorylation on tyrosine 14 is linked to augmented melanoma metastasis but not tumorigenesis.

ABSTRACT: Caveolin-1 (CAV1) is a scaffolding protein that plays a dual role in cancer. In advanced stages of this disease, CAV1 expression in tumor cells is associated with enhanced metastatic potential, while, at earlier stages, CAV1 functions as a tumor suppressor. We recently implicated CAV1 phosphorylation on tyrosine 14 (Y14) in CAV1-enhanced cell migration. However, the contribution of this modification to the dual role of CAV1 in cancer remained unexplored. Here, we used in vitro [2D and transendothelial cell migration (TEM), invasion] and in vivo (metastasis) assays, as well as genetic and biochemical approaches to address this question in B16F10 murine melanoma cells. CAV1 promoted directional migration on fibronectin or laminin, two abundant lung extracellular matrix (ECM) components, which correlated with enhanced Y14 phosphorylation during spreading. Moreover, CAV1-driven migration, invasion, TEM and metastasis were ablated by expression of the phosphorylation null CAV1(Y14F), but not the phosphorylation mimicking CAV1(Y14E) mutation. Finally, CAV1-enhanced focal adhesion dynamics and surface expression of beta1 integrin were required for CAV1-driven TEM. Importantly, CAV1 function as a tumor suppressor in tumor formation assays was not altered by the Y14F mutation. In conclusion, our results provide critical insight to the mechanisms of CAV1 action during cancer development. Specific ECM-integrin interactions and Y14 phosphorylation are required for CAV1-enhanced melanoma cell migration, invasion and metastasis to the lung. Because Y14F mutation diminishes metastasis without inhibiting the tumor suppressor function of CAV1, Y14 phosphorylation emerges as an attractive therapeutic target to prevent metastasis without altering beneficial traits of CAV1.

SUBMITTER: Ortiz R

PROVIDER: S-EPMC5130029 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Extracellular matrix-specific Caveolin-1 phosphorylation on tyrosine 14 is linked to augmented melanoma metastasis but not tumorigenesis.

Ortiz Rina R Díaz Jorge J Díaz Natalia N Lobos-Gonzalez Lorena L Cárdenas Areli A Contreras Pamela P Díaz María Inés MI Otte Ellen E Cooper-White Justin J Torres Vicente V Leyton Lisette L Quest Andrew F G AF

Oncotarget 20160601 26

Caveolin-1 (CAV1) is a scaffolding protein that plays a dual role in cancer. In advanced stages of this disease, CAV1 expression in tumor cells is associated with enhanced metastatic potential, while, at earlier stages, CAV1 functions as a tumor suppressor. We recently implicated CAV1 phosphorylation on tyrosine 14 (Y14) in CAV1-enhanced cell migration. However, the contribution of this modification to the dual role of CAV1 in cancer remained unexplored. Here, we used in vitro [2D and transendot ...[more]

PMID: 27259249

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Project description:Phosphorylated tyrosine hydroxylase (TH) can form complexes with 14-3-3 proteins, resulting in enzyme activation and stabilization. Although TH was among the first binding partners identified for these ubiquitous regulatory proteins, the binding stoichiometry and the activation mechanism remain unknown. To address this, we performed native mass spectrometry analyses of human TH (nonphosphorylated or phosphorylated on Ser19 (TH-pS19), Ser40 (TH-pS40), or Ser19 and Ser40 (TH-pS19pS40)) alone and together with 14-3-3?. Tetrameric TH-pS19 (224 kDa) bound 14-3-3? (58.3 kDa) with high affinity (Kd = 3.2 nM), generating complexes containing either one (282.4 kDa) or two (340.8 kDa) dimers of 14-3-3. Electron microscopy also revealed one major population of an asymmetric complex, consistent with one TH tetramer and one 14-3-3 dimer, and a minor population of a symmetric complex of one TH tetramer with two 14-3-3 dimers. Lower phosphorylation stoichiometries (0.15-0.54 phosphate/monomer) produced moderate changes in binding kinetics, but native MS detected much less of the symmetric TH:14-3-3? complex. Interestingly, dephosphorylation of [(32)P]-TH-pS19 was mono-exponential for low phosphorylation stoichiometries (0.18-0.52), and addition of phosphatase accelerated the dissociation of the TH-pS19:14-3-3? complex 3- to 4-fold. All together this is consistent with a model in which the pS19 residues in the TH tetramer contribute differently in the association to 14-3-3?. Complex formation between TH-pS40 and 14-3-3? was not detected via native MS, and surface plasmon resonance showed that the interaction was very weak. Furthermore, TH-pS19pS40 behaved similarly to TH-pS19 in terms of binding stoichiometry and affinity (Kd = 2.1 nM). However, we found that 14-3-3? inhibited the phosphorylation rate of TH-pS19 by PKA (3.5-fold) on Ser40. We therefore conclude that Ser40 does not significantly contribute to the binding of 14-3-3?, and rather has reduced accessibility in the TH:14-3-3? complex. This adds to our understanding of the fine-tuned physiological regulation of TH, including hierarchical phosphorylation at multiple sites.

Dataset Information

Extracellular matrix-specific Caveolin-1 phosphorylation on tyrosine 14 is linked to augmented melanoma metastasis but not tumorigenesis.

Publications

Extracellular matrix-specific Caveolin-1 phosphorylation on tyrosine 14 is linked to augmented melanoma metastasis but not tumorigenesis.

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