Phosphorylation-dependent recognition of diverse protein targets by the cryptic GK domain of MAGI MAGUKs.

Dynamic signal transduction requires the rapid assembly and disassembly of signaling complexes, often mediated by phosphoprotein binding modules. The guanylate kinase-like (GK) domain of the membrane-associated guanylate kinases (MAGUKs) is such a module orchestrating signaling at cellular junctions. The MAGI subfamily of MAGUKs contains a truncated GK domain with unknown structure and function, although they participate in diverse physiological and pathological processes. Here, we demonstrate that the truncated GK domain of MAGI2 interacts with its adjacent PDZ0 domain to form a structural supramodule capable of recognizing phosphoproteins. A conserved phosphorylation-dependent binding motif for PDZ0-GK is delineated, which leads to identification of a set of previously unknown binding partners. We explore the structure and function of the MAGI2-target complex with an inhibitory peptide derived from the consensus motif. Our work reveals an action mechanism of the cryptic MAGI GKs and broadens our understanding of the target recognition rules of phosphoprotein binding modules.

Structural Analysis Implicates CASK-Liprin-α2 Interaction in Cerebellar Granular Cell Death in MICPCH Syndrome.

Microcephaly with pontine and cerebellar hypoplasia (MICPCH) syndrome is a neurodevelopmental disorder caused by the deficiency of the X-chromosomal gene CASK. However, the molecular mechanisms by which CASK deficiency causes cerebellar hypoplasia in this syndrome remain elusive. In this study, we used CASK knockout (KO) mice as models for MICPCH syndrome and investigated the effect of CASK mutants. Female CASK heterozygote KO mice replicate the progressive cerebellar hypoplasia observed in MICPCH syndrome. CASK KO cultured cerebellar granule (CG) cells show progressive cell death that can be rescued by co-infection with lentivirus expressing wild-type CASK. Rescue experiments with CASK deletion mutants identify that the CaMK, PDZ, and SH3, but not L27 and guanylate kinase domains of CASK are required for the survival of CG cells. We identify missense mutations in the CaMK domain of CASK derived from human patients that fail to rescue the cell death of cultured CASK KO CG cells. Machine learning-based structural analysis using AlphaFold 2.2 predicts that these mutations disrupt the structure of the binding interface with Liprin-α2. These results suggest that the interaction with Liprin-α2 via the CaMK domain of CASK may be involved in the pathophysiology of cerebellar hypoplasia in MICPCH syndrome.

A novel missense variant in the CASK gene causes intellectual developmental disorder and microcephaly with pontine and cerebellar hypoplasia.

BACKGROUND: Variants in the CASK gene result in a wide range of observed phenotypes in humans, such as FG Syndrome 4 and intellectual disabilities. Intellectual developmental disorder with microcephaly and pontine and cerebellar hypoplasia (MICPCH) is an X-linked disorder that affects females and is characterized by severely impaired intellectual development and variable degrees of pontocerebellar hypoplasia. Variants in CASK are the main genetic cause of MICPCH. Variants in CASK can explain most patients with MICPCH, but there are still some patients whose disease aetiology cannot be explained. CASE PRESENTATION: An 11-month-old female diagnosed with MICPCH exhibited general developmental delays, microcephaly, and cerebellar hypoplasia. Whole-exome sequencing (WES) was used to find a novel heterozygous missense variant (NM_003688.3: c.638T>G) of CASK in this patient. Strikingly, this variant reduced the expression of CASK at the protein level but not at the mRNA level. By using protein structure prediction analysis, this study found that the amino acid change caused by the variant resulted in further changes in the stability of the protein structure, and these changes caused the downregulation of protein expression and loss of protein function. CONCLUSION: In this study, we first reported a novel heterozygous pathogenic variant and a causative mechanism of MICPCH. The amino acid change cause by this variant led to changes in the protein structure and a decrease in its stability, which caused a loss of protein function. This study could be helpful to the genetic diagnosis of this disease.

A de novo variant in CASK gene causing intellectual disability and brain hypoplasia: a case report and literature review.

BACKGROUND: The pathogenic variation of CASK gene can cause CASK related mental disorders. The main clinical manifestations are microcephaly with pontine and cerebellar hypoplasia, X-linked mental disorders with or without nystagmus and FG syndrome. The main pathogenic mechanism is the loss of function of related protein caused by variant. We reported a Chinese male newborn with a de novo variant in CASK gene. CASE PRESENTATION: We present an 18-day-old baby with growth retardation and brain hypoplasia. Whole-exome sequencing was performed, which detected a hemizygous missense variant c.764G > A of CASK gene. The variant changed the 255th amino acid from Arg to His. Software based bioinformatics analyses were conducted to infer its functional effect. CONCLUSIONS: In this paper, a de novo variant of CASK gene was reported. Moreover, a detailed description of all the cases described in the literature is reported. CASK variants cause a variety of clinical phenotypes. Its diagnosis is difficult due to the lack of typical clinical symptoms. Genetic testing should be performed as early as possible if this disease is suspected. This case provides an important reference for the diagnosis and treatment of future cases.

Reactivities of the Front Pocket N-Terminal Cap Cysteines in Human Kinases.

The front pocket (FP) N-terminal cap (Ncap) cysteine is the most popular site of covalent modification in kinases. A long-standing hypothesis associates the Ncap position with cysteine hyper-reactivity; however, traditional computational predictions suggest that the FP Ncap cysteines are predominantly unreactive. Here we applied the state-of-the-art continuous constant pH molecular dynamics (CpHMD) to test the Ncap hypothesis. Simulations found that the Ncap cysteines of BTK/BMX/TEC/ITK/TXK, JAK3, and MKK7 are reactive to varying degrees; however, those of BLK and EGFR/ERBB2/ERBB4 possessing a Ncap+3 aspartate are unreactive. Analysis suggested that hydrogen bonding and electrostatic interactions drive the reactivity, and their absence renders the Ncap cysteine unreactive. To further test the Ncap hypothesis, we examined the FP Ncap+2 cysteines in JNK1/JNK2/JNK3 and CASK. Our work offers a systematic understanding of the cysteine structure-reactivity relationship and illustrates the use of CpHMD to differentiate cysteines toward the design of targeted covalent inhibitors with reduced chemical reactivities.

Exploration of the Hsa-miR-1587-Protein Interaction and the Inhibition to CASK.

Hsa-miR-1587 has been found to be capable of forming G-quadruplex structures and is overexpressed in multiple cancer cell lines. Here, we explored the interactions between miR-1587 and proteins. HuProt™ human proteome microarray was utilized to screen the binding proteins, and it was discovered that CASK could bind to miR-1587 on the base of the G-quadruplex structure. Moreover, reelin and p21, which are downstream of CASK, were downregulated both transcriptionally and translationally by miR-1587, uncovered by q-RT-PCR and Western blot assays. Bioinformatic analysis was performed on STRING and Panther platforms, leading to the discovery that miR-1587 may be involved in intracellular metabolic and transcriptional physiological processes. This study explores the interaction of hsa-miR-1587 with proteins and provides a new strategy for the regulation of G-rich microRNA's function.

Crystal structure of the PDZ4 domain of MAGI2 in complex with PBM of ARMS reveals a canonical PDZ recognition mode.

Membrane-associated guanylate kinase, WW and PDZ domain-containing protein 2 (MAGI2) is a neuronal scaffold protein that plays critical roles at synaptic junctions by assembling neurotransmitter receptors and cell adhesion proteins through its multiple protein-protein interaction domains, including six PDZ domains, two phosphoserine-phosphothreonine binding WW domains, and a guanylate kinase GK domain. Previous studies showed that MAGI2 participates in formation of tetrameric complexes with PDZ-GEF1, TrkA receptor, and ankyrin repeat-rich membrane spanning (ARMS) protein at late endosomes and is crucial for neurite outgrowth. However, the molecular mechanism governing the assembly of these complexes remains unknown. Here, we characterize the direct interaction between MAGI2 and ARMS through multiple biochemical assays. Moreover, our solved crystal structure of the truncated PDZ4/PBM (PDZ binding motifs) complex of MAGI2 and ARMS proteins (MAGI2-PDZ4/ARMS-PBM) reveals that the binding interface lies between the αB/ßB groove from the PDZ4 of MAGI2 and the C-terminal PBM from ARMS. The structure reveals high similarity to others in this protein family where canonical PDZ/PBM interactions are observed. However, the conserved "GLGF" motif in the PSD-95-PDZ3 changes to "GFGF" in the MAGI2-PDZ4/ARMS-PBM complex. We further validated our crystal structure through serial mutagenesis assays. Taken together, our study provides the biochemical details and binding mechanisms that underpin the stabilization of the MAGI2-PDZ4/ARMS-PBM complex, thereby offering a biochemical and structural basis for further understanding of the functional roles of MAGI2, ARMS, PDZ-GEF1, and TrkA in forming the tetrameric receptor complex in neuronal signaling.

Phase Separation of MAGI2-Mediated Complex Underlies Formation of Slit Diaphragm Complex in Glomerular Filtration Barrier.

BACKGROUND: Slit diaphragm is a specialized adhesion junction between the opposing podocytes, establishing the final filtration barrier to urinary protein loss. At the cytoplasmic insertion site of each slit diaphragm there is an electron-dense and protein-rich cellular compartment that is essential for slit diaphragm integrity and signal transduction. Mutations in genes that encode components of this membrane-less compartment have been associated with glomerular diseases. However, the molecular mechanism governing formation of compartmentalized slit diaphragm assembly remains elusive. METHODS: We systematically investigated the interactions between key components at slit diaphragm, such as MAGI2, Dendrin, and CD2AP, through a combination of biochemical, biophysical, and cell biologic approaches. RESULTS: We demonstrated that MAGI2, a unique MAGUK family scaffold protein at slit diaphragm, can autonomously undergo liquid-liquid phase separation. Multivalent interactions among the MAGI2-Dendrin-CD2AP complex drive the formation of the highly dense slit diaphragm condensates at physiologic conditions. The reconstituted slit diaphragm condensates can effectively recruit Nephrin. A nephrotic syndrome-associated mutation of MAGI2 interfered with formation of the slit diaphragm condensates, thus leading to impaired enrichment of Nephrin. CONCLUSIONS: Key components at slit diaphragm (e.g., MAGI2 and its complex) can spontaneously undergo phase separation. The reconstituted slit diaphragm condensates can be enriched in adhesion molecules and cytoskeletal adaptor proteins. Therefore, the electron-dense slit diaphragm assembly might form via phase separation of core components of the slit diaphragm in podocytes.

Functional analysis of CASK transcript variants expressed in human brain.

The calcium-/calmodulin dependent serine protein kinase (CASK) belongs to the membrane-associated guanylate kinases (MAGUK) family of proteins. It fulfils several different cellular functions, ranging from acting as a scaffold protein to transcription control, as well as regulation of receptor sorting. CASK functions depend on the interaction with a variety of partners, for example neurexin, liprin-α, Tbr1 and SAP97. So far, it is uncertain how these seemingly unrelated interactions and resulting functions of CASK are regulated. Here, we show that alternative splicing of CASK can guide the binding affinity of CASK isoforms to distinct interaction partners. We report seven different variants of CASK expressed in the fetal human brain. Four out of these variants are not present in the NCBI GenBank database as known human variants. Functional analyses showed that alternative splicing affected the affinities of CASK variants for several of the tested interaction partners. Thus, we observed a clear correlation of the presence of one splice insert with poor binding of CASK to SAP97, supported by molecular modelling. The alternative splicing and distinct properties of CASK variants in terms of protein-protein interaction should be taken into consideration for future studies.

An ancestral MAGUK protein supports the modulation of mammalian voltage-gated Ca2+ channels through a conserved CaVß-like interface.

Eukaryote voltage-gated Ca2+ channels of the CaV2 channel family are hetero-oligomers formed by the pore-forming CaVα1 protein assembled with auxiliary CaVα2δ and CaVß subunits. CaVß subunits are formed by a Src homology 3 (SH3) domain and a guanylate kinase (GK) domain connected through a HOOK domain. The GK domain binds a conserved cytoplasmic region of the pore-forming CaVα1 subunit referred as the "AID". Herein we explored the phylogenetic and functional relationship between CaV channel subunits in distant eukaryotic organisms by investigating the function of a MAGUK protein (XM_004990081) cloned from the choanoflagellate Salpingoeca rosetta (Sro). This MAGUK protein (Sroß) features SH3 and GK structural domains with a 25% primary sequence identity to mammalian CaVß. Recombinant expression of its cDNA with mammalian high-voltage activated Ca2+ channel CaV2.3 in mammalian HEK cells produced robust voltage-gated inward Ca2+ currents with typical activation and inactivation properties. Like CaVß, Sroß prevents fast degradation of total CaV2.3 proteins in cycloheximide assays. The three-dimensional homology model predicts an interaction between the GK domain of Sroß and the AID motif of the pore-forming CaVα1 protein. Substitution of AID residues Trp (W386A) and Tyr (Y383A) significantly impaired co-immunoprecipitation of CaV2.3 with Sroß and functional upregulation of CaV2.3 currents. Likewise, a 6-residue deletion within the GK domain of Sroß, similar to the locus found in mammalian CaVß, significantly reduced peak current density. Altogether our data demonstrate that an ancestor MAGUK protein reconstitutes the biophysical and molecular features responsible for channel upregulation by mammalian CaVß through a minimally conserved molecular interface.

Structural Basis for the High-Affinity Interaction between CASK and Mint1.

CASK forms an evolutionarily conserved tripartite complex with Mint1 and Veli critical for neuronal synaptic transmission and cell polarity. The CASK CaM kinase (CaMK) domain, in addition to interacting with Mint1, can also bind to many different target proteins, although the mechanism governing CASK-CaMK/target interaction selectivity is unclear. Here, we demonstrate that an extended sequence in the N-terminal unstructured region of Mint1 binds to CASK-CaMK with a dissociation constant of ∼7.5 nM. The high-resolution crystal structure of CASK-CaMK in complex with this Mint1 fragment reveals that the C-lobe of CASK-CaMK binds to a short sequence common to known CaMK targets and the N-lobe of CaMK engages an α helix that is unique to Mint1. Biochemical experiments together with structural analysis reveal that the CASK and Mint1 interaction is not regulated by Ca2+/CaM. The CASK/Mint1 complex structure provides mechanistic explanations for several CASK mutations identified in patients with brain disorders and cancers.

Distinct calcium/calmodulin-dependent serine protein kinase domains control cardiac sodium channel membrane expression and focal adhesion anchoring.

BACKGROUND: Membrane-associated guanylate kinase proteins function as adaptor proteins to mediate the recruitment and scaffolding of ion channels in the plasma membrane in various cell types. In the heart, the protein calcium/calmodulin-dependent serine protein kinase (CASK) negatively regulates the main cardiac sodium channel NaV1.5, which carries the sodium current (INa) by preventing its anterograde trafficking. CASK is also a new member of the dystrophin-glycoprotein complex and, like syntrophin, binds to the C-terminal domain of the channel. OBJECTIVE: The purpose of this study was to unravel the mechanisms of CASK-mediated negative INa regulation and interaction with the dystrophin-glycoprotein complex in cardiac myocytes. METHODS: CASK adenoviral truncated constructs with sequential single functional domain deletions were designed for overexpression in cardiac myocytes: CASKΔCAMKII, CASKΔL27A, CASKΔL27B, CASKΔPDZ, CASKΔSH3, CASKΔHOOK, and CASKΔGUK. A combination of whole-cell patch-clamp recording, total internal reflection fluorescence microscopy, and biochemistry experiments was conducted in cardiac myocytes to study the functional consequences of domain deletions. RESULTS: We show that both L27B and GUK domains are required for the negative regulatory effect of CASK on INa and NaV1.5 surface expression and that the HOOK domain is essential for interaction with the cell adhesion dystrophin-glycoprotein complex. CONCLUSION: This study demonstrates that the multimodular structure of CASK confers an ability to simultaneously interact with several targets within cardiomyocytes. Through its L27B, GUK, and HOOK domains, CASK potentially provides the ability to control channel delivery at adhesion points in cardiomyocytes.

Acquisition of a phospho-acceptor site enhances HPV E6 PDZ-binding motif functional promiscuity.

All cancer-causing human papillomavirus (HPV) E6 oncoproteins have a C-terminal PDZ-binding motif (PBM), which correlates with oncogenic potential. Nonetheless, several HPVs with little or no oncogenic potential also have an E6 PBM, with minor sequence differences affecting PDZ protein selectivity. Furthermore, certain HPV types have a phospho-acceptor site embedded within the PBM. We therefore compared HPV-18, HPV-66 and HPV-40 E6 proteins to examine the possible link between the ability to target multiple PDZ proteins and the acquisition of a phospho-acceptor site. The mutation of essential residues in HPV-18E6 reduces its phosphorylation, and fewer PDZ substrates are bound. In contrast, the generation of consensus phospho-acceptor sites in HPV-66 and HPV-40 E6 PBMs increases the PDZ proteins recognized. Thus, although phosphorylation of the E6 PBM and PDZ protein recognition are mutually exclusive, they are closely linked, with the acquisition of a phospho-acceptor site also contributing to an expansion in the number of PDZ proteins bound.

The polarity protein Dlg5 regulates collective cell migration during Drosophila oogenesis.

Collective migration plays critical roles in animal development, physiological events, and cancer metastasis. However, the molecular mechanisms of collective cell migration are not well understood. Drosophila border cells represent an excellent in vivo genetic model to study collective cell migration and identify novel regulatory genes for cell migration. Using the Mosaic Analysis with a Repressible Cell Marker (MARCM) system, we screened 240 P-element insertion lines to identify essential genes for border cell migration. Two genes were uncovered, including dlg5 (discs large 5) and CG31689. Further analysis showed that Dlg5 regulates the apical-basal polarity and cluster integrity in border cell clusters. Dlg5 is enriched in lateral surfaces between border cells and central polar cells but also shows punctate localization between border cells. We found that the distribution of Dlg5 in border cell clusters is regulated by Armadillo. Structure-function analysis revealed that the N-terminal Coiled-coil domain and the C-terminal PDZ3-PDZ4-SH3-GUK domains but not the PDZ1-PDZ2 domains of Dlg5 are required for BC migration. The Coiled-coil domain and the PDZ4-SH3-GUK domains are critical for Dlg5's cell surface localization in border cell clusters.

Structural determinants governing ß-arrestin2 interaction with PDZ proteins and recruitment to CRFR1.

ß-Arrestins are multifunctional adaptor proteins best know for their vital role in regulating G protein coupled receptor (GPCR) trafficking and signaling. ß-arrestin2 recruitment and receptor internalization of corticotropin-releasing factor receptor 1 (CRFR1), a GPCR whose antagonists have been shown to demonstrate both anxiolytic- and antidepressant-like effects, have previously been shown to be modulated by PDZ proteins. Thus, a structural characterization of the interaction between ß-arrestins and PDZ proteins can delineate potential mechanism of PDZ-dependent regulation of GPCR trafficking. Here, we find that the PDZ proteins PSD-95, MAGI1, and PDZK1 interact with ß-arrestin2 in a PDZ domain-dependent manner. Further investigation of such interaction using mutational analyses revealed that mutating the alanine residue at 175 residue of ß-arrestin2 to phenylalanine impairs interaction with PSD-95. Additionally, A175F mutant of ß-arrestin2 shows decreased CRF-stimulated recruitment to CRFR1 and reduced receptor internalization. Thus, our findings show that the interaction between ß-arrestins and PDZ proteins is key for CRFR1 trafficking and may be targeted to mitigate impaired CRFR1 signaling in mental and psychiatric disorders.

Solution structure and functional investigation of human guanylate kinase reveals allosteric networking and a crucial role for the enzyme in cancer.

Human guanylate kinase (hGMPK) is the only known enzyme responsible for cellular GDP production, making it essential for cellular viability and proliferation. Moreover, hGMPK has been assigned a critical role in metabolic activation of antiviral and antineoplastic nucleoside-analog prodrugs. Given that hGMPK is indispensable for producing the nucleotide building blocks of DNA, RNA, and cGMP and that cancer cells possess elevated GTP levels, it is surprising that a detailed structural and functional characterization of hGMPK is lacking. Here, we present the first high-resolution structure of hGMPK in the apo form, determined with NMR spectroscopy. The structure revealed that hGMPK consists of three distinct regions designated as the LID, GMP-binding (GMP-BD), and CORE domains and is in an open configuration that is nucleotide binding-competent. We also demonstrate that nonsynonymous single-nucleotide variants (nsSNVs) of the hGMPK CORE domain distant from the nucleotide-binding site of this domain modulate enzymatic activity without significantly affecting hGMPK's structure. Finally, we show that knocking down the hGMPK gene in lung adenocarcinoma cell lines decreases cellular viability, proliferation, and clonogenic potential while not altering the proliferation of immortalized, noncancerous human peripheral airway cells. Taken together, our results provide an important step toward establishing hGMPK as a potential biomolecular target, from both an orthosteric (ligand-binding sites) and allosteric (location of CORE domain-located nsSNVs) standpoint.

An N-terminal heterozygous missense CASK mutation is associated with microcephaly and bilateral retinal dystrophy plus optic nerve atrophy.

Heterozygous loss-of-function mutations in the X-linked gene CASK are associated with mental retardation and microcephaly with pontine and cerebellar hypoplasia (MICPCH) and ophthalmological disorders including optic nerve atrophy (ONA) and optic nerve hypoplasia (ONH). Recently, we have demonstrated that CASK(+/-) mice display ONH with 100% penetrance but exhibit no change in retinal lamination or structure. It is not clear if CASK loss-of-function predominantly affects retinal ganglion cells, or if other retinal cells like photoreceptors are also involved. Here, we report a heterozygous missense mutation in the N-terminal calcium/calmodulin-dependent kinase (CaMK) domain of the CASK protein in which a highly conserved leucine is mutated to the cyclic amino acid proline. In silico analysis suggests that the mutation may produce destabilizing structural changes. Experimentally, we observe pronounced misfolding and insolubility of the CASKL209P protein. Interestingly, the remaining soluble mutant protein fails to interact with Mint1, which specifically binds to CASK's CaMK domain, suggesting a mechanism for the phenotypes observed with the CASKL209P mutation. In addition to microcephaly, cerebellar hypoplasia and delayed development, the subject with the L209P mutation also presented with bilateral retinal dystrophy and ONA. Electroretinography indicated that rod photoreceptors are the most prominently affected cells. Our data suggest that the CASK interactions mediated by the CaMK domain may play a crucial role in retinal function, and thus, in addition to ONH, individuals with mutations in the CASK gene may exhibit other retinal disorders, depending on the nature of mutation.

Gain-of-Function Mutation of Card14 Leads to Spontaneous Psoriasis-like Skin Inflammation through Enhanced Keratinocyte Response to IL-17A.

Genetic mutations of CARD14 (encoding CARMA2) are observed in psoriasis patients. Here we showed that Card14E138A/+ and Card14ΔQ136/+ mice developed spontaneous psoriasis-like skin inflammation, which resulted from constitutively activated CARMA2 via self-aggregation leading to the enhanced activation of the IL-23-IL-17A cytokine axis. Card14-/- mice displayed attenuated skin inflammation in the imiquimod-induced psoriasis model due to impaired IL-17A signaling in keratinocytes. CARMA2, mainly expressed in keratinocytes, associates with the ACT1-TRAF6 signaling complex and mediates IL-17A-induced NF-κB and MAPK signaling pathway activation, which leads to expression of pro-inflammatory factors. Thus, CARMA2 serves as a key mediator of IL-17A signaling and its constitutive activation in keratinocytes leads to the onset of psoriasis, which indicates an important role of NF-κB activation in keratinocytes in psoriatic initiation.

Dissipation at the angstrom scale: Probing the surface and interior of an enzyme.

Pursuing a materials science approach to understanding the deformability of enzymes, we introduce measurements of the phase of the mechanical response function within the nanorheology paradigm. Driven conformational motion of the enzyme is dissipative as characterized by the phase measurements. The dissipation originates both from the surface hydration layer and the interior of the molecule, probed by examining the effect of point mutations on the mechanics. We also document changes in the mechanics of the enzyme examined, guanylate kinase, upon binding its four substrates. GMP binding stiffens the molecule, ATP and ADP binding softens it, while there is no clear mechanical signature of GDP binding. A hyperactive two-Gly mutant is found to possibly trade specificity for speed. Global deformations of enzymes are shown to be dependent on both hydration layer and polypeptide chain dynamics.

Two microcephaly-associated novel missense mutations in CASK specifically disrupt the CASK-neurexin interaction.

Deletion and truncation mutations in the X-linked gene CASK are associated with severe intellectual disability (ID), microcephaly and pontine and cerebellar hypoplasia in girls (MICPCH). The molecular origin of CASK-linked MICPCH is presumed to be due to disruption of the CASK-Tbr-1 interaction. This hypothesis, however, has not been directly tested. Missense variants in CASK are typically asymptomatic in girls. We report three severely affected girls with heterozygous CASK missense mutations (M519T (2), G659D (1)) who exhibit ID, microcephaly, and hindbrain hypoplasia. The mutation M519T results in the replacement of an evolutionarily invariant methionine located in the PDZ signaling domain known to be critical for the CASK-neurexin interaction. CASKM519T is incapable of binding to neurexin, suggesting a critically important role for the CASK-neurexin interaction. The mutation G659D is in the SH3 (Src homology 3) domain of CASK, replacing a semi-conserved glycine with aspartate. We demonstrate that the CASKG659D mutation affects the CASK protein in two independent ways: (1) it increases the protein's propensity to aggregate; and (2) it disrupts the interface between CASK's PDZ (PSD95, Dlg, ZO-1) and SH3 domains, inhibiting the CASK-neurexin interaction despite residing outside of the domain deemed critical for neurexin interaction. Since heterozygosity of other aggregation-inducing mutations (e.g., CASKW919R) does not produce MICPCH, we suggest that the G659D mutation produces microcephaly by disrupting the CASK-neurexin interaction. Our results suggest that disruption of the CASK-neurexin interaction, not the CASK-Tbr-1 interaction, produces microcephaly and cerebellar hypoplasia. These findings underscore the importance of functional validation for variant classification.