Dataset Information

Crk adaptor proteins are necessary for the development of the zebrafish retina.

ABSTRACT:

Background

The development of the central nervous system (CNS) requires critical cell signaling molecules to coordinate cell proliferation and migration in order to structure the adult tissue. Chicken tumor virus #10 Regulator of Kinase (CRK) and CRK-like (CRKL) are adaptor proteins with pre-metazoan ancestry and are known to be required for patterning laminated structures downstream of Reelin (RELN), such as the cerebral cortex, cerebellum, and hippocampus. CRK and CRKL also play crucial roles in a variety of other growth factor and extracellular matrix signaling cascades. The neuronal retina is another highly laminated structure within the CNS that is dependent on migration for proper development, but the cell signaling mechanisms behind neuronal positioning in the retina are only partly understood.

Results

We find that crk and crkl have largely overlapping expression within the developing zebrafish nervous system. We find that their disruption results in smaller eye size and loss of retinal lamination.

Conclusions

Our data indicate that Crk adaptors are critical for proper development of the zebrafish neural retina in a crk/crkl dose-dependent manner.

SUBMITTER: Stergas HR

PROVIDER: S-EPMC8760358 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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Publications

Crk adaptor proteins are necessary for the development of the zebrafish retina.

Stergas Helaina R HR Kalbag Zoë Z St Clair Riley M RM Talbot Jared C JC Ballif Bryan A BA Ebert Alicia M AM

Developmental dynamics : an official publication of the American Association of Anatomists 20210724 2

<h4>Background</h4>The development of the central nervous system (CNS) requires critical cell signaling molecules to coordinate cell proliferation and migration in order to structure the adult tissue. Chicken tumor virus #10 Regulator of Kinase (CRK) and CRK-like (CRKL) are adaptor proteins with pre-metazoan ancestry and are known to be required for patterning laminated structures downstream of Reelin (RELN), such as the cerebral cortex, cerebellum, and hippocampus. CRK and CRKL also play crucia ...[more]

PMID: 34268820

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Project description:IntroductionCT10 regulator of kinase (Crk) adaptor proteins (CrkI, CrkII and CrkL) play a role in integrating signals for migration and invasion of highly malignant breast cancer cell lines. This has important implications, as elevated CrkI/II protein levels were observed in a small cohort of breast cancer patients, which identified a potential role for Crk proteins in breast cancer progression. Numerous in vitro studies identified a role for Crk proteins in cell motility, but little is known about how Crk proteins contribute to breast cancer progression in vivo.MethodsThe clinical significance of Crk proteins in human breast cancer was assessed by analyzing published breast cancer datasets using a gene expression signature that was generated following CrkII over-expression and by examining Crk protein expression in tissue microarrays of breast tumors (n = 254). Stable knockdown of Crk (CrkI/CrkII/CrkL) proteins was accomplished using a short hairpin RNA (shRNA)-mediated approach in two basal breast cancer cell lines, MDA-231 1833TR and SUM1315, where the former have a high affinity to form bone metastases. Both in vitro assays (cell migration, invasion, soft agar growth) and in vivo experiments (intra-cardiac, tibial and mammary fat pad injections) were performed to assess the functional significance of Crk proteins in breast cancer.ResultsA gene signature derived following CrkII over-expression correlated significantly with basal breast cancers and with high grade and poor outcome in general. Moreover, elevated Crk immunostaining on tissue microarrays revealed a significant association with highly proliferative tumors within the basal subtype. RNAi-mediated knockdown of all three Crk proteins in metastatic basal breast cancer cells established a continued requirement for Crk in cell migration and invasion in vitro and metastatic growth in vivo. Furthermore, Crk ablation suppressed anchorage independent growth and in vivo orthotopic tumor growth. This was associated with diminished cell proliferation and was rescued by expression of non-shRNA targeted CrkI/II. Perturbations in tumor progression correlated with altered integrin signaling, including decreased cell spreading, diminished p130Cas phosphorylation, and Cdc42 activation.ConclusionsThese data highlight the physiological importance of Crk proteins in regulating growth of aggressive basal breast cancer cells and identify Crk-dependent signaling networks as promising therapeutic targets.

Project description:BackgroundC3G is a guanine nucleotide exchange factor (GEF) that activates Rap1 to promote cell adhesion. Resting C3G is autoinhibited and the GEF activity is released by stimuli that signal through tyrosine kinases. C3G is activated by tyrosine phosphorylation and interaction with Crk adaptor proteins, whose expression is elevated in multiple human cancers. However, the molecular details of C3G activation and the interplay between phosphorylation and Crk interaction are poorly understood.MethodsWe combined biochemical, biophysical, and cell biology approaches to elucidate the mechanisms of C3G activation. Binding of Crk adaptor proteins to four proline-rich motifs (P1 to P4) in C3G was characterized in vitro using isothermal titration calorimetry and sedimentation velocity, and in Jurkat and HEK293T cells by affinity pull-down assays. The nucleotide exchange activity of C3G over Rap1 was measured using nucleotide-dissociation kinetic assays. Jurkat cells were also used to analyze C3G translocation to the plasma membrane and the C3G-dependent activation of Rap1 upon ligation of T cell receptors.ResultsCrkL interacts through its SH3N domain with sites P1 and P2 of inactive C3G in vitro and in Jurkat and HEK293T cells, and these sites are necessary to recruit C3G to the plasma membrane. However, direct stimulation of the GEF activity requires binding of Crk proteins to the P3 and P4 sites. P3 is occluded in resting C3G and is essential for activation, while P4 contributes secondarily towards complete stimulation. Tyrosine phosphorylation of C3G alone causes marginal activation. Instead, phosphorylation primes C3G lowering the concentration of Crk proteins required for activation and increasing the maximum activity. Unexpectedly, optimal activation also requires the interaction of CrkL-SH2 domain with phosphorylated C3G.ConclusionOur study revealed that phosphorylation of C3G by Src and Crk-binding form a two-factor mechanism that ensures tight control of C3G activation. Additionally, the simultaneous SH2 and SH3N interaction of CrkL with C3G, required for the activation, reveals a novel adaptor-independent function of Crk proteins relevant to understanding their role in physiological signaling and their deregulation in diseases. Video abstract.

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Dataset Information

Crk adaptor proteins are necessary for the development of the zebrafish retina.

Background

Results

Conclusions

Publications

Crk adaptor proteins are necessary for the development of the zebrafish retina.

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