CRKL but not CRKII contributes to hemin-induced erythroid differentiation of CML.

Destruction of erythropoiesis process leads to various diseases, including thrombocytopenia, anaemia, and leukaemia. miR-429-CT10 regulation of kinase-like (CRKL) axis involved in development, progression and metastasis of cancers. However, the exact role of miR-429-CRKL axis in leukaemic cell differentiation are still unknown. The current work aimed to uncover the effect of miR-429-CRKL axis on erythropoiesis. In the present study, CRKL upregulation was negatively correlated with miR-429 downregulation in both chronic myeloid leukaemia (CML) patient and CR patient samples. Moreover, CRKL expression level was significantly decreased while miR-429 expression level was increased during the erythroid differentiation of K562 cells following hemin treatment. Functional investigations revealed that overexpression and knockdown of CRKL was remarkably effective in suppressing and promoting hemin-induced erythroid differentiation of K562 cells, whereas, miR-429 exhibited opposite effects to CRKL. Mechanistically, miR-429 regulates erythroid differentiation of K562 cells by downregulating CRKL via selectively targeting CRKL-3'-untranslated region (UTR) through Raf/MEK/ERK pathway. Conversely, CRKII had no effect on erythroid differentiation of K562 cells. Taken together, our data demonstrated that CRKL (but not CRKII) and miR-429 contribute to development, progression and erythropoiesis of CML, miR-429-CRKL axis regulates erythropoiesis of K562 cells via Raf/MEK/ERK pathway, providing novel insights into effective diagnosis and therapy for CML patients.

Identification of CRKL as an oncogenic biomarker for prognosis and immunotherapy in melanoma, and its potential molecular mechanism.

CRKL (CRK Like Proto-Oncogene) belongs to the Crk family and is a 39-kDa adapter protein that encodes SH2 and SH3 (src homologs) domains. To identify its oncogenic role in malignant melanoma, we investigated the association between CRKL and mutation, prognosis, tumor mutation burden, immune cell infiltration of melanoma, and explored the associations between CRKL and immunotherapy response. Our results showed that abnormal CRKL expression is associated with poor prognosis in melanoma and is significantly correlated with immune-activated pathways and processes, immune cell infiltrations, and expression of immunoregulators. Importantly, we found that CRKL expression is a predictive biomarker for anti-PD1 therapy response in melanoma patients. Furthermore, inhibiting CRKL expression in melanoma cell lines suppressed their proliferation and metastasis, as well as activated the pyroptosis-related pathway. Our study provides potential mechanisms of melanoma pathogenesis, which may suggest new avenues for targeted therapy in this disease.

Gab2 plays a carcinogenic role in ovarian cancer by regulating CrkII.

OBJECTIVE: To detect the expression of Growth factor binding protein 2 associated binding protein 2 (Gab2) and CT10 regulator of kinase II (CrkII) in ovarian cancer and analyze their clinical significance. To explore the effects of Gab2 and CrkII on the biological behavior of ovarian cancer cells. To analyze the possible molecular mechanism of Gab2 in the development of ovarian cancer. METHODS: Immunohistochemistry was used to detect the expression of Gab2 and CrkII in ovarian cancer. Chi square test was used to analyze the correlation between Gab2, CrkII and clinical parameters. Using Cox regression model to evaluate the risk factors affecting the prognosis. To analyze the correlation between Gab2, CrkII and survival rate by Kaplan-Meier. Cell experiments were preformed to explore the effects of Gab2 and CrkII on the biological behavior of cells. The interaction between Gab2 and CrkII was explored by immunoprecipitation. RESULTS: Immunohistochemistry revealed that high expression of Gab2 and CrkII in ovarian cancer. Patients with high expression of Gab2 or CrkII had higher International Federation of Gynecology and Obstetrics (FIGO) stage, grade and platinum-resistance recurrence. Multivariate analysis showed that Gab2 and CrkII were independent prognostic factors. Kaplan-Meier curve showed that the higher Gab2 and CrkII were, the poor prognosis the patients had. We observed that the overexpression of Gab2 and CrkII promoted the proliferation, metastasis and reduced chemosensitivity of cells. Conversely, the knockdown of Gab2 and CrkII resulted in the opposite results. In CrkII-knockdown cells, we found that Gab2 mediates biological behavior through CrkII. CONCLUSIONS: The expression of Gab2 and CrkII increase in ovarian cancer. The higher expression of Gab2 and CrkII predict the poor prognosis of patients. Gab2 and CrkII promote the proliferation and migration and reduce the chemosensitivity of cells. Gab2 regulates the biological behaviors of ovarian cancer cells through CrkII.

Crk proteins activate the Rap1 guanine nucleotide exchange factor C3G by segregated adaptor-dependent and -independent mechanisms.

BACKGROUND: C3G 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. METHODS: We 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. RESULTS: CrkL 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. CONCLUSION: Our 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.

Control of CRK-RAC1 activity by the miR-1/206/133 miRNA family is essential for neuromuscular junction function.

Formation and maintenance of neuromuscular junctions (NMJs) are essential for skeletal muscle function, allowing voluntary movements and maintenance of the muscle tone, thereby preventing atrophy. Generation of NMJs depends on the interaction of motor neurons with skeletal muscle fibers, which initiates a cascade of regulatory events that is essential for patterning of acetylcholine receptor (AChR) clusters at specific sites of the sarcolemma. Here, we show that muscle-specific miRNAs of the miR-1/206/133 family are crucial regulators of a signaling cascade comprising DOK7-CRK-RAC1, which is critical for stabilization and anchoring of postsynaptic AChRs during NMJ development and maintenance. We describe that posttranscriptional repression of CRK by miR-1/206/133 is essential for balanced activation of RAC1. Failure to adjust RAC1 activity severely compromises NMJ function, causing respiratory failure in neonates and neuromuscular symptoms in adult mice. We conclude that miR-1/206/133 serve a specific function for NMJs but are dispensable for skeletal muscle development.

Crk and Crkl have shared functions in neural crest cells for cardiac outflow tract septation and vascular smooth muscle differentiation.

CRK and CRKL encode cytoplasmic adaptors that contribute to the etiology of congenital heart disease. Neural crest cells (NCCs) are required for cardiac outflow tract (OFT) septation and aortic arch formation. The roles of Crk/Crkl in NCCs during mouse cardiovascular development remain unknown. To test this, we inactivated Crk and/or Crkl in NCCs. We found that the loss of Crk, rather than Crkl, in NCCs resulted in double outlet right ventricle, while loss of both Crk/Crkl in NCCs resulted in severe defects with earlier lethality due to failed OFT septation and severe dilation of the pharyngeal arch arteries (PAAs). We found that these defects are due to altered cell morphology resulting in reduced localization of NCCs to the OFT and failed integrity of the PAAs, along with reduced expression of Integrin signaling genes. Further, molecular studies identified reduced differentiation of vascular smooth muscle cells that may in part be due to altered Notch signaling. Additionally, there is increased cellular stress that leads to modest increase in apoptosis. Overall, this explains the mechanism for the Crk/Crkl phenotype.

Silencing of lncRNA MEG8 Represses the Viability, Migration, and Invasion of Wilms' Tumor Cells through Mediating miR-23a-3p/CRK Axis.

PURPOSE: Compelling evidence has unveiled the importance of long noncoding RNAs (lncRNAs) in malignant behavior of Wilms' tumor (WT). Hereon, we intend to assess the function and associated molecular mechanism of lncRNA maternally expressed gene 8 (MEG8) in WT cells. METHODS: Expression levels of MEG8, miR-23a-3p, and CT10 regulator of kinase (CRK) were determined by quantitative real-time polymerase chain reaction. Cell viability was assessed by MTT assay. Besides, wound healing assay and transwell assay were applied to examine abilities of cell migration and invasion, respectively. Dual-luciferase reporter assay was employed to test the interplay among MEG8, miR-23a-3p, and CRK. Western blot was used to detect relative protein expression of CRK. RESULTS: MEG8 and CRK expression was elevated, while miR-23a-3p expression was decreased in WT tissues and cells. The histologic type, lymphatic metastasis, and National Wilms Tumor Study (NWTS) stage were associated with the expression of MEG8, miR-23a-3p, and CRK in WT patients. MEG8 knockdown or miR-23a-3p overexpression restrained WT cells in cell viability, migration, and invasiveness in vitro. As to mechanism exploration, MEG8 could directly bind to miR-23a-3p and then miR-23a-3p targeted CRK. MEG8 was inversely correlated with miR-23a-3p and positively correlated with CRK in WT tissues. Meantime, miR-23a-3p was inversely correlated with CRK in WT tissues. Additionally, MEG8 knockdown-mediated suppressive impacts on cell viability, migration, and invasiveness were reversed by overexpression of CRK or repression of miR-23a-3p in WT cells. CONCLUSIONS: The cell viability, migration, and invasiveness of WT cells were repressed by MEG8 knockdown via targeting the miR-23a-3p/CRK axis.

CD93 Signaling via Rho Proteins Drives Cytoskeletal Remodeling in Spreading Endothelial Cells.

During angiogenesis, cell adhesion molecules expressed on the endothelial cell surface promote the growth and survival of newly forming vessels. Hence, elucidation of the signaling pathways activated by cell-to-matrix adhesion may assist in the discovery of new targets to be used in antiangiogenic therapy. In proliferating endothelial cells, the single-pass transmembrane glycoprotein CD93 has recently emerged as an important endothelial cell adhesion molecule regulating vascular maturation. In this study, we unveil a signaling pathway triggered by CD93 that regulates actin cytoskeletal dynamics responsible of endothelial cell adhesion. We show that the Src-dependent phosphorylation of CD93 and the adaptor protein Cbl leads to the recruitment of Crk, which works as a downstream integrator in the CD93-mediated signaling. Moreover, confocal microscopy analysis of FRET-based biosensors shows that CD93 drives the coordinated activation of Rac1 and RhoA at the cell edge of spreading cells, thus promoting the establishment of cell polarity and adhesion required for cell motility.

MicroRNA-215-5p Inhibits the Proliferation and Migration of Wilm's Tumor Cells by Targeting CRK.

OBJECTIVE: Wilm's tumor is a common renal malignancy in childhood with unsatisfactory prognosis. microRNA-215-5p (miR-215-5p) has been reported as a tumor-suppressive miRNA in different types of human cancers, but rarely in the Wilm's tumor. In light of this, we tried to investigate the regulatory role and underlying mechanism of miR-215-5p in the Wilm's tumor. METHODS: After sample collection and cell culture, the expression of miR-215-5p and CT10 Regulator of Kinase (CRK) was detected. Then rhabdoid tumor cell lines (formerly classified as Wilms' tumor cell lines), G401 and WT-CLS1 cells were transfected with pcDNA3.1, pcDNA3.1-CRK, sh-NC, sh-CRK, agomir NC, miR-215-5p agomir, antagomir NC or miR-215-5p antagomir to explore the function of miR-215-5p and CRK in the Wilm's tumor cell proliferation and migration. Moreover, the relationship between miR-215-5p and CRK was analyzed by dual luciferase reporter gene assay. RESULTS: Lowly-expressed miR-215-5p and highly-expressed CRK were observed in the Wilm's tumor tissues and cells. Transfection of pcDNA3.1-CRK or miR-215-5p antagomir could promote G401 and WT-CLS1 cell proliferation and enhance migration ability, while transfection of sh-CRK or miR-215-5p agomir led to opposite results. Additionally, miR-215-5p may bind to CRK. Moreover, transfection of pcDNA3.1-CRK in G401 and WT-CLS1 cells could partially reverse the inhibitory effect of miR-215-5p agomir on the proliferation and migration of Wilm's tumor cells. CONCLUSION: Our study highlighted that miR-215-5p could suppress the proliferation and migration of Wilm's tumor cells by regulating the expression of CRK, providing new ideas for molecular targeted therapy for Wilm's tumor.

Pyk2 regulates cell-edge protrusion dynamics by interacting with Crk.

Focal adhesion kinase (FAK) is well established as a regulator of cell migration, but whether and how the closely related proline-rich tyrosine kinase 2 (Pyk2) regulates fibroblast motility is still under debate. Using mouse embryonic fibroblasts (MEFs) from Pyk2-/- mice, we show here, for the first time, that lack of Pyk2 significantly impairs both random and directed fibroblast motility. Pyk2-/- MEFs show reduced cell-edge protrusion dynamics, which is dependent on both the kinase and protein-protein binding activities of Pyk2. Using bioinformatics analysis of in vitro high- throughput screens followed by text mining, we identified CrkI/II as novel substrates and interactors of Pyk2. Knockdown of CrkI/II shows altered dynamics of cell-edge protrusions, which is similar to the phenotype observed in Pyk2-/- MEFs. Moreover, epistasis experiments suggest that Pyk2 regulates the dynamics of cell-edge protrusions via direct and indirect interactions with Crk that enable both activation and down-regulation of Crk-mediated cytoskeletal signaling. This complex mechanism may enable fine-tuning of cell-edge protrusion dynamics and consequent cell migration on the one hand together with tight regulation of cell motility, a process that should be strictly limited to specific time and context in normal cells, on the other hand.

Mechanism of disease and therapeutic rescue of Dok7 congenital myasthenia.

Congenital myasthenia (CM) is a devastating neuromuscular disease, and mutations in DOK7, an adaptor protein that is crucial for forming and maintaining neuromuscular synapses, are a major cause of CM1,2. The most common disease-causing mutation (DOK71124_1127 dup) truncates DOK7 and leads to the loss of two tyrosine residues that are phosphorylated and recruit CRK proteins, which are important for anchoring acetylcholine receptors at synapses. Here we describe a mouse model of this common form of CM (Dok7CM mice) and a mouse with point mutations in the two tyrosine residues (Dok72YF). We show that Dok7CM mice had severe deficits in neuromuscular synapse formation that caused neonatal lethality. Unexpectedly, these deficits were due to a severe deficiency in phosphorylation and activation of muscle-specific kinase (MUSK) rather than a deficiency in DOK7 tyrosine phosphorylation. We developed agonist antibodies against MUSK and show that these antibodies restored neuromuscular synapse formation and prevented neonatal lethality and late-onset disease in Dok7CM mice. These findings identify an unexpected cause for disease and a potential therapy for both DOK7 CM and other forms of CM caused by mutations in AGRIN, LRP4 or MUSK, and illustrate the potential of targeted therapy to rescue congenital lethality.

Crk and CrkL as Therapeutic Targets for Cancer Treatment.

Crk and CrkL are cellular counterparts of the viral oncoprotein v-Crk. Crk and CrkL are overexpressed in many types of human cancer, correlating with poor prognosis. Furthermore, gene knockdown and knockout of Crk and CrkL in tumor cell lines suppress tumor cell functions, including cell proliferation, transformation, migration, invasion, epithelial-mesenchymal transition, resistance to chemotherapy drugs, and in vivo tumor growth and metastasis. Conversely, overexpression of tumor cells with Crk or CrkL enhances tumor cell functions. Therefore, Crk and CrkL have been proposed as therapeutic targets for cancer treatment. However, it is unclear whether Crk and CrkL make distinct or overlapping contributions to tumor cell functions in various cancer types because Crk or CrkL have been examined independently in most studies. Two recent studies using colorectal cancer and glioblastoma cells clearly demonstrated that Crk and CrkL need to be ablated individually and combined to understand distinct and overlapping roles of the two proteins in cancer. A comprehensive understanding of individual and overlapping roles of Crk and CrkL in tumor cell functions is necessary to develop effective therapeutic strategies. This review systematically discusses crucial functions of Crk and CrkL in tumor cell functions and provides new perspectives on targeting Crk and CrkL in cancer therapy.

Cell shape-based chemical screening reveals an epigenetic network mediated by focal adhesions.

Adapter proteins CRK and CRKL participate in a variety of signaling pathways, including cell adhesion, and fate regulation of mammalian cells. However, the molecular functions of CRK/CRKL in epigenetic regulation remain largely unknown. Here, we developed a pipeline to evaluate cell morphology using high-content image analysis combined with chemical screening of kinase and epigenetic modulators. We found that CRK/CRKL modulates gene regulatory networks associated with cell morphology through epigenetic alteration in mouse embryonic fibroblasts. Integrated epigenome and transcriptome analyses revealed that CRK/CRKL is involved in super-enhancer activity and upregulation of Cdt1, Rin1, and Spp1 expression for the regulation of cell morphology. Screening of a library of 80 epigenetic inhibitors showed that histone H3 modifiers, euchromatic histone methyltransferase 2 and mitogen- and stress-activated kinase 1, may be important for CRK/CRKL-mediated morphological changes. Taken together, our results indicate that CRK/CRKL plays a critical role in gene regulatory networks through epigenetic modification. DATABASES: Chromatin immunoprecipitation sequencing and RNA sequencing data were deposited in the DNA Data Bank of Japan under DRA011080 and DRA011081 accession numbers, respectively.

Quantitative assessment of glioblastoma phenotypes in vitro establishes cell migration as a robust readout of Crk and CrkL activity.

The expression levels of CT10 regulator of kinase (Crk) and Crk-like (CrkL) are elevated in many human cancers, including glioblastoma (GBM), and are believed to contribute to poor prognosis. Although Crk and CrkL have been proposed as therapeutic targets in these tumors, the lack of a reliable, quantitative assay to measure Crk and CrkL activity has hindered development of inhibitors. Here, we knocked down Crk, CrkL, or both using siRNAs in a human GBM cell line, U-118MG, to determine the respective, quantitative contributions of Crk and CrkL to cellular phenotypes. The combined use of specific and potent Crk and CrkL siRNAs induced effective knockdown of CrkII, CrkI, and CrkL. Whereas Crk knockdown did not affect cell morphology, proliferation, adhesion, or invasion, CrkL knockdown caused shrinkage of cells and inhibition of cell proliferation, adhesion, and invasion. Crk/CrkL double knockdown resulted in more pronounced morphological alterations and more robust inhibition of proliferation, adhesion, and invasion. Furthermore, Crk/CrkL double knockdown completely blocked cell migration, and this effect was rescued by transient overexpression of CrkL but not of Crk. Quantification of protein levels indicated that CrkL is expressed more abundantly than CrkII and CrkI in U-118MG cells. These results demonstrate both the predominant role of CrkL and the essential overlapping functions of Crk and CrkL in U-118MG cells. Furthermore, our study indicates that migration of U-118MG cells depends entirely on Crk and CrkL. Thus, impedance-based, real-time measurement of tumor cell migration represents a robust assay for monitoring Crk and CrkL activities.

Genome-Wide Identification and Characterization of Calcium-Dependent Protein Kinase (CDPK) and CDPK-Related Kinase (CRK) Gene Families in Medicago truncatula.

Calcium-dependent protein kinase (CDPK or CPK) and CDPK-related kinase (CRK) play an important role in plant growth, development, and adaptation to environmental stresses. However, their gene families had been yet inadequately investigated in Medicago truncatula. In this study, six MtCRK genes were computationally identified, they were classified into five groups with MtCDPKs based on phylogenetic relationships. Six pairs of segmental duplications were observed in MtCDPK and MtCRK genes and the Ka/Ks ratio, an indicator of selection pressure, was below 0.310, indicating that these gene pairs underwent strong purifying selection. Cis-acting elements of morphogenesis, multiple hormone responses, and abiotic stresses were predicted in the promoter region. The spatial expression of MtCDPKs and MtCRKs displays diversity. The expression of MtCDPKs and MtCRKs could be regulated by various stresses. MtCDPK4, 14, 16, 22, and MtCRK6 harbor both N-myristoylation site and palmitoylation site and were anchored on plasma membrane, while MtCDPK7, 9, and 15 contain no or only one N-acylation site and were distributed in cytosol and nucleus, suggesting that the N-terminal acylation sites play a key role in subcellular localization of MtCDPKs and MtCRKs. In summary, comprehensive characterization of MtCDPKs and MtCRKs provide a subset of candidate genes for further functional analysis and genetic improvement against drought, cold, salt and biotic stress.

Novel CRK-Cyclin Complex Controls Spindle Assembly Checkpoint in Toxoplasma Endodyogeny.

Opportunistic parasites of the Apicomplexa phylum use a variety of division modes built on two types of cell cycles that incorporate two distinctive mechanisms of mitosis: uncoupled from and coupled to parasite budding. Parasites have evolved novel factors to regulate such unique replication mechanisms that are poorly understood. Here, we have combined genetics, quantitative fluorescence microscopy, and global proteomics approaches to examine endodyogeny in Toxoplasma gondii dividing by mitosis coupled to cytokinesis. In the current study, we focus on the steps controlled by the recently described atypical Cdk-related kinase T. gondii Crk6 (TgCrk6). While inspecting protein complexes, we found that this previously orphaned TgCrk6 kinase interacts with a parasite-specific atypical cyclin, TgCyc1. We built conditional expression models and examined primary cell cycle defects caused by the lack of TgCrk6 or TgCyc1. Quantitative microscopy assays revealed that tachyzoites deficient in either TgCrk6 or the cyclin partner TgCyc1 exhibit identical mitotic defects, suggesting cooperative action of the complex components. Further examination of the mitotic structures indicated that the TgCrk6/TgCyc1 complex regulates metaphase. This novel finding confirms a functional spindle assembly checkpoint (SAC) in T. gondii. Measuring global changes in protein expression and phosphorylation, we found evidence that canonical activities of the Toxoplasma SAC are intertwined with parasite-specific tasks. Analysis of phosphorylation motifs suggests that Toxoplasma metaphase is regulated by CDK, mitogen-activated kinase (MAPK), and Aurora kinases, while the TgCrk6/TgCyc1 complex specifically controls the centromere-associated network. IMPORTANCE The rate of Toxoplasma tachyzoite division directly correlates with the severity of the disease, toxoplasmosis, which affects humans and animals. Thus, a better understanding of the tachyzoite cell cycle would offer much-needed efficient tools to control the acute stage of infection. Although tachyzoites divide by binary division, the cell cycle architecture and regulation differ significantly from the conventional binary fission of their host cells. Unlike the unidirectional conventional cell cycle, the Toxoplasma budding cycle is braided and is regulated by multiple essential Cdk-related kinases (Crks) that emerged in the place of missing conventional cell cycle regulators. How these novel Crks control apicomplexan cell cycles is largely unknown. Here, we have discovered a novel parasite-specific complex, TgCrk6/TgCyc1, that orchestrates a major mitotic event, the spindle assembly checkpoint. We demonstrated that tachyzoites incorporated parasite-specific tasks in the canonical checkpoint functions.

Crk Haploinsufficiency Is Associated with Intrauterine Growth Retardation and Severe Postnatal Growth Failure.

BACKGROUND: Children with 17p13.3 microdeletions including the YWHAE gene show intrauterine growth restriction, craniofacial dysmorphisms, postnatal growth failure, and cognitive impairment. This region is characterized by genomic instability and has been associated with isolated lissencephaly sequence and Miller-Dieker syndrome characterized by facial dysmorphisms, microcephaly, short stature, seizures, cardiac malformations, and agyria. Whilst brain abnormalities are secondary to YWHAE deficiency, the cause of pre- and postnatal growth failure has not been identified yet. CASE PRESENTATION: We describe 2 patients (patient 1 15 years and patient 2 11 years and 10 months) referred to our Center of Pediatric Endocrinology for intrauterine growth retardation with de novo 17p13.3 deletion. In vitro assays showed a defect in CRK expression and GH/IGF1 signaling. rhGH therapy was effective in partially reducing the deficit in height in patient 1 and induced catch-up growth in patient 2. CONCLUSION: Our results suggest that 17p13.3 microdeletion involving CRK affects both GH and IGF1 signaling ultimately leading to pre- and postnatal growth retardation, secondary to partial insensitivity to GH/IGF1. rhGH therapy may be considered to reduce the height deficit in these patients, though data on adult height are lacking.

Prenatal diagnosis of Miller-Dieker syndrome by chromosomal microarray.

OBJECTIVE: To assess the experience on prenatal diagnosis of Miller-Dieker syndrome (MDS) to further delineate the fetal presentation of this syndrome. METHODS: This was a retrospective study. Fetal MDS was diagnosed prenatally by chromosomal microarray (CMA). Clinical data were reviewed for these cases, including maternal characteristics, indications for prenatal diagnosis, sonographic findings, CMA results, and pregnancy outcomes. RESULTS: Four cases were diagnosis as MDS by CMA. The most common sonographic features were ventriculomegaly (3/4) and polyhydramnios (2/4). Deletion sizes ranged from 1.5 to 5.4 Mb. All microdeletions were located at the MDS critical region and showed haploinsufficiency of the YWHAE, CRK, and PAFAH1B1. All patients chose to terminate the pregnancy. Parental chromosome analysis were preformed in three cases and demonstrated that two cases were de novo and one case was caused by inherited derivative chromosomes from parental balanced translocations. CONCLUSION: The most common prenatal ultrasound findings of MDS were ventriculomegaly and polyhydramnios. CMA can improve diagnostic precision for detecting MDS.

Requirement for Crk and CrkL during postnatal lens development.

The Crk and CrkL adaptor proteins have SH2 and SH3 domains and play essential overlapping, as well as distinct, roles in many biological processes, ranging from cell structure and motility to proliferation. Conditional ablation of both Crk and CrkL in neuronal progenitor cells, using a Nestin-Cre transgene, resulted in severe defects in postnatal eye development, including progressive eye closure, lens rupture, and retinal malformation. These phenotypes were not observed in the presence of a single wild-type allele of either Crk or CrkL. We found that the lens in knockout mice started to rupture and disintegrate between postnatal days 7 and 12, although the structure of the retina was relatively well maintained. As the lens deteriorated further, the outer nuclear layer in the posterior of the retina enlarged and developed ruffles. Cre recombination occurred in the lens and retina of the knockout mice. Furthermore, the posterior lens capsule of the knockout mouse was thinner at postnatal days 0.5 and 3, suggesting that the defective lens capsule caused rupturing of the lens near the posterior pole. These results indicate that Crk and CrkL play essential overlapping roles in postnatal lens development.

Crk1/2 and CrkL play critical roles in maintaining podocyte morphology and function.

Podocytes are actin-rich epithelial cells whose effacement and detachment are the main cause of glomerular disease. Crk family proteins: Crk1/2 and CrkL are reported to be important intracellular signaling proteins that are involved in many biological processes. However, the roles of them in maintaining podocyte morphology and function remain poorly understood. In this study, specific knocking down of Crk1/2 and CrkL in podocytes caused abnormal cell morphology, actin cytoskeleton rearrangement and dysfunction in cell adhesion, spreading, migration, and viability. The p130Cas, focal adhesion kinase, phosphatidylinositol 3-kinase/Akt, p38 and JNK signaling pathways involved in these alterations. Furthermore, knocking down CrkL alone conferred a more modest phenotype than did the Crk1/2 knockdown and the double knockdown. Kidney biopsy specimens from patients with focal segmental glomerulosclerosis and minimal change nephropathy showed downregulation of Crk1/2 and CrkL in glomeruli. In zebrafish embryos, Crk1/2 and CrkL knockdown compromised the morphology and caused abnormal glomerular development. Thus, our results suggest that Crk1/2 and CrkL expression are important in podocytes; loss of either will cause podocyte dysfunction, leading to foot process effacement and podocyte detachment.