The cytoplasmic domains of UNC5 are responsible for its netrin-mediated signaling events in axonal migrations, blood vessel patterning, and apoptosis, although the molecular mechanisms governing these processes are unknown. To provide a foundation for the elucidation of the UNC5-mediated signaling mechanism, we determined the crystal structure of the cytoplasmic portion of UNC5b. We found that it contains three distinctly folded domains, namely ZU5, UPA, and death domain (DD). These three domains form a structural supramodule, with ZU5 binding to both UPA and DD, thereby locking the ZU5-UPA-DD supramodule in a closed conformation and suppressing its biological activities. Release of the closed conformation of the ZU5-UPA-DD supramodule leads to the activation of the receptor in the promotion of apoptosis and blood vessel patterning. Finally, we provide evidence showing that the supramodular nature of UNC5 ZU5-UPA-DD is likely to be shared by the ankyrin and PIDD families of scaffold proteins.

Myosin VI is the only known molecular motor that moves toward the minus ends of actin filaments; thus, it plays unique roles in diverse cellular processes. The processive walking of myosin VI on actin filaments requires dimerization of the motor, but the protein can also function as a nonprocessive monomer. The molecular mechanism governing the monomer-dimer conversion is not clear. We report the high-resolution NMR structure of the cargo-free myosin VI cargo-binding domain (CBD) and show that it is a stable monomer in solution. The myosin VI CBD binds to a fragment of the clathrin-coated vesicle adaptor Dab2 with a high affinity, and the X-ray structure of the myosin VI CBD in complex with Dab2 reveals that the motor undergoes a cargo-binding-mediated dimerization. The cargo-binding-induced dimerization may represent a general paradigm for the regulation of processivity for myosin VI as well as other myosins, including myosin VII and myosin X.

Summary Asymmetric cell division requires the establishment of cortical cell polarity and the orientation of the mitotic spindle along the axis of cell polarity. Evidence from invertebrates demonstrates that the Par3/Par6/aPKC and NuMA/LGN/Gαi complexes, which are thought to be physically linked by the adapter protein mInscuteable (mInsc), play indispensable roles in this process. However, the molecular basis for the binding of LGN to NuMA and mInsc is poorly understood. The high resolution structures of the LGN/NuMA and LGN/mInsc complexes presented here provide mechanistic insights into the distinct and highly specific interactions of the LGN TPR repeats with mInsc and NuMA. Structural comparisons, together with biochemical and cell biology studies, demonstrate that the interactions of NuMA and mInsc with LGN are mutually exclusive, with mInsc binding preferentially. Our results suggest that the Par3/mInsc/LGN and NuMA/LGN/Gαi complexes play sequential and partially overlapping roles in asymmetric cell division.

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Membrane-associated guanylate kinases (MAGUKs) are a large family of scaffold proteins that play essential roles in tissue developments, cell-cell communications, cell polarity control, and cellular signal transductions. Despite extensive studies over the past two decades, the functions of the signature guanylate kinase domain (GK) of MAGUKs are poorly understood. Here we show that the GK domain of DLG1/SAP97 binds to asymmetric cell division regulatory protein LGN in a phosphorylation-dependent manner. The structure of the DLG1 SH3-GK tandem in complex with a phospho-LGN peptide reveals that the GMP-binding site of GK has evolved into a specific pSer/ pThr-binding pocket. Residues both N-and C-terminal to the pSer are also critical for the specific binding of the phospho-LGN peptide to GK. We further demonstrate that the previously reported GK domain-mediated interactions of DLGs with other targets, such as GKAP/DLGAP1/ SAPAP1 and SPAR, are also phosphorylation dependent. Finally, we provide evidence that other MAGUK GKs also function as phospho-peptide-binding modules. The discovery of the phosphorylation-dependent MAGUK GK/ target interactions indicates that MAGUK scaffoldmediated signalling complex organizations are dynamically regulated.

INAD is a scaffolding protein that regulates signaling in Drosophila photoreceptors. One of its PDZ domains, PDZ5, cycles between reduced and oxidized forms in response to light, but it is unclear how light affects its redox potential. Through biochemical and structural studies, we show that the redox potential of PDZ5 is allosterically regulated by its interaction with another INAD domain, PDZ4. Whereas isolated PDZ5 is stable in the oxidized state, formation of a PDZ45 "supramodule" locks PDZ5 in the reduced state by raising the redox potential of its Cys606/Cys645 disulfide bond by ∼330 mV. Acidification, potentially mediated via light and PLCβ-mediated hydrolysis of PIP(2), disrupts the interaction between PDZ4 and PDZ5, leading to PDZ5 oxidation and dissociation from the TRP Ca(2+) channel, a key component of fly visual signaling. These results show that scaffolding proteins can actively modulate the intrinsic redox potentials of their disulfide bonds to exert regulatory roles in signaling.

Dynein light chain 1 (DLC1, also known as DYNLL1, LC8, and PIN), a ubiquitously expressed and highly conserved protein, participates in a variety of essential intracellular events. Transition of DLC1 between dimer and monomer forms might play a crucial role in its function. However, the molecular mechanism(s) that control the transition remain unknown. DLC1 phosphorylation on Ser 88 by p21-activated kinase 1 (Pak1), a signaling nodule, promotes mammalian cell survival by regulating its interaction with Bim and the stability of Bim. Here we discovered that phosphorylation of Ser 88 , which juxtapose each other at the interface of the DLC dimer, disrupts DLC1 dimer formation and consequently impairs its interaction with Bim. Overexpression of a Ser 88 phosphorylation-inactive DLC1 mutant in mammary epithelium cells and in a transgenic animal model caused apoptosis and accelerated mammary gland involution, respectively, with increased Bim levels. Structural and biophysical studies suggested that phosphorylation-mimicking mutation leads to dissociation of the DLC1 dimer to a pure folded monomer. The phosphorylation-induced DLC1 monomer is incapable of binding to its substrate Bim. These findings reveal a previously unrecognized regulatory mechanism of DLC1 in which the Ser 88 phosphorylation acts as a molecular switch for the transition of DLC1 from dimer to monomer, thereby modulating its interaction with substrates and consequently regulating the functions of DLC1.Dyneins are massive minus-to-plus end microtubules motor complexes. Dyneins are categorized into axonemal dyneins and cytoplasmic dyneins on the basis of structural and functional features (1). Cytoplasmic dyneins are essential for a variety of fundamental intracellular events, such as organization and orientation of the mitotic spindle, nuclear migration, Golgi dynamics, retrograde neuronal axonal transport, and trafficking of vesicles and molecules (2-4). Dynein light chain 1 (DLC1) 4 (also known as DYNLL1, LC8, DLC8, and PIN), a ubiquitously expressed 89-amino acid protein, was initially identified as a light chain of the Chlamydomonas outer dynein arm (5). DLC1 is highly conserved from nematodes to mammals, and DLC1 orthologues share more than 90% sequence identity (6). DLC1 binds to a diverse array of proteins and RNAs, including neuronal nitric-oxide synthase (7), IB␣ (8), p53-binding protein 1 (9), GKAP (10), gephyrin postsynaptic scaffolding proteins (11), Bim (12), Swallow (13), estrogen receptor (14), KIBRA (15), CDK2 (16), virus proteins (17, 18), parathyroid hormone mRNA (19), and possibly other proteins (20, 21).Although DLC1 has been shown to bind to many partners, its physiological roles and the upstream regulators of DLC1 remain poorly understood. DLC1 is presumed to have essential cell functions because of its extraordinary sequence conservation across species and ubiquitous expression. Genetic studies in Aspergillus suggested that DLC1 is important for activities of dynein, because a DLC1 temperature-sensitive mutation led to multiple d...

Pleckstrin homology (PH) domains play diverse roles in cytoskeletal dynamics and signal transduction. Split PH domains represent a unique subclass of PH domains that have been implicated in interactions with complementary partial PH domains 'hidden' in many proteins. Whether partial PH domains exist as independent structural units alone and whether two halves of a split PH domain can fold together to form an intact PH domain are not known. Here, we solved the structure of the PH N -PDZ-PH C tandem of a-syntrophin. The split PH domain of a-syntrophin adopts a canonical PH domain fold. The isolated partial PH domains of a-syntrophin, although completely unfolded, remain soluble in solution. Mixing of the two isolated domains induces de novo folding and yields a stable PH domain. Our results demonstrate that two complementary partial PH domains are capable of binding to each other to form an intact PH domain. We further showed that the PH N -PDZ-PH C tandem forms a functionally distinct supramodule, in which the split PH domain and the PDZ domain function synergistically in binding to inositol phospholipids.