*2.6. Noonan Syndrome*

Noonan syndrome (NS) is a complex disease where patients present various clinical features including short stature, dysmorphia, and di fferent cardiac defects [71,72]. Infants a ffected by the disease su ffer from polyhydramnios, pleural e ffusions, or edema. Recent studies have linked NS to lung lymphangiectasis that is characterized by vessel dilatation and hemorrhages. NS is caused by germline mutations in genes that encode protein components of the RAS/mitogen activated protein kinase (MAPK) pathway. The vast majority of these mutations result in increased RAS/MAPK signaling [75].

Germline mutations of leucine zipper-like transcriptional regulator 1 (LZTR1) are associated with Noonan syndrome and familial schwannomatosis [71,109] (Table 1). Schwannomatosis is characterized by multiple 'schwannomas', benign tumors of the peripheral and spinal nerves [110]. *Lztr1* loss in mice results in perinatal lethality due to cardiovascular dysfunction [12,70]. *Lztr1* haploinsu fficiency in mice partially recapitulates NS phenotypes, including heart malformation, craniofacial features, and bleeding abnormalities [70]. *LZTR1* loss in Schwann cells promotes their proliferation and dedi fferentiation [12].

LZTR1 serves as a substrate adaptor for Cullin3 (CUL3) ubiquitin ligase complex that controls ubiquitination of the RAS GTPases [12,30,111]. LZTR1-mediated monoubiquitination of RAS at K170 attenuates RAS association with the membrane, inhibiting the RAS signaling pathway [12,30]. Disease-associated *LZTR1* mutations diminish either LZTR1-CUL3 complex formation or its interaction with RAS proteins, which impairs RAS ubiquitination. Dysregulation of RAS monoubiquitination results in the hyperactivation of the MAPK pathway and explains the role of LZTR1 in NS and schwannomatosis (Figure 1D). LZTR1 was also reported to regulate vesicular tra fficking of VEGFR through CHMP1B ubiquitination. Loss of *Lztr1* reduces multi-monoubiquitination of CHMP1B, thus blocking the disassembly of the ESCRT complex and the tra fficking of endosomal VEGFR. This ultimately leads to the activation of VEGF signaling and explains the bleeding abnormalities detected in NS patients [70].

The RING-type E3 ligase Casitas B-lineage lymphoma (CBL) is also found to be mutated in Noonan syndrome-like disorder, as well as early-onset juvenile myelomonocytic leukemia (JMML) (Table 1). JMML is a myeloproliferative disorder characterized by malignant transformation of the hematopoietic stem cell (HSC) causing oncogenic transformation of HSC-derived cells. Most common *CBL* mutations in patients a ffect the RING domains, such as the Y371 residue, as its phosphorylation is important for activation of the ligase function [76,77]. Deletion of *c-Cbl* in mice causes a decrease of B- and T-lymphocyte function causing lymphopenia [112], partially recapitulating JMML. *c-Cbl* knockout mice also present increased sensitivity to cardiac infarct [113], echoing the cardiovascular defects detected in NS patients.

CBL is a key regulator of internalization and turnover of di fferent receptor tyrosine kinases, as it monoubiquitinates endosomal adaptors important for the recycling and/or degradation of the RTKs [114]. Loss of CBL results in endosomal accumulation of the RTK and uncontrolled activation of the downstream pathways [73]. One of the endosomal adaptors modified by CBL is SH3 domain-containing kinase-binding protein 1 (SH3KBP1, also called CIN85), which undergoes ubiquitination in response to EGF stimulation. After EGFR is internalized, monoubiquitinated SH3KBP1 recruits active EGFR to the multivesicular bodies (MVB) that targets EGFR for degradation. Loss of *CBL* leads to constitutive activation of EGF signaling [74]. The alterations in the tra fficking and degradation of these signaling proteins lead to up-regulation of the MAPK pathway that could at least partially explain the mechanism by which CBL contributes to human disease (Figure 1E). Interestingly, deletion of the *SH3KBP1* gene in B-cells in mice results in impaired T-cell-independent antibody responses and abnormal B-cell receptor response, partially recapitulating JMML phenotypes [115].
