Characterization of the SAM domain of the PKD-related protein ANKS6 and its interaction with ANKS3.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic disorder leading to end-stage renal failure in humans. In the PKD/Mhm(cy/+) rat model of ADPKD, the point mutation R823W in the sterile alpha motif (SAM) domain of the protein ANKS6 is responsible for disease. SAM domains are known protein-protein interaction domains, capable of binding each other to form polymers and heterodimers. Despite its physiological importance, little is known about the function of ANKS6 and how the R823W point mutation leads to PKD. Recent work has revealed that ANKS6 interacts with a related protein called ANKS3. Both ANKS6 and ANKS3 have a similar domain structure, with ankyrin repeats at the N-terminus and a SAM domain at the C-terminus. RESULTS: The SAM domain of ANKS3 is identified as a direct binding partner of the ANKS6 SAM domain. We find that ANKS3-SAM polymerizes and ANKS6-SAM can bind to one end of the polymer. We present crystal structures of both the ANKS3-SAM polymer and the ANKS3-SAM/ANKS6-SAM complex, revealing the molecular details of their association. We also learn how the R823W mutation disrupts ANKS6 function by dramatically destabilizing the SAM domain such that the interaction with ANKS3-SAM is lost. CONCLUSIONS: ANKS3 is a direct interacting partner of ANKS6. By structurally and biochemically characterizing the interaction between the ANKS3 and ANKS6 SAM domains, our work provides a basis for future investigation of how the interaction between these proteins mediates kidney function.

Tandem SAM domain structure of human Caskin1: a presynaptic, self-assembling scaffold for CASK.

The synaptic scaffolding proteins CASK and Caskin1 are part of the fibrous mesh of proteins that organize the active zones of neural synapses. CASK binds to a region of Caskin1 called the CASK interaction domain (CID). Adjacent to the CID, Caskin1 contains two tandem sterile α motif (SAM) domains. Many SAM domains form polymers so they are good candidates for forming the fibrous structures seen in the active zone. We show here that the SAM domains of Caskin1 form a new type of SAM helical polymer. The Caskin1 polymer interface exhibits a remarkable segregation of charged residues, resulting in a high sensitivity to ionic strength in vitro. The Caskin1 polymers can be decorated with CASK proteins, illustrating how these proteins may work together to organize the cytomatrix in active zones.

A human sterile alpha motif domain polymerizome.

The sterile alpha motif (SAM) domain is one of the most common protein modules found in eukaryotic genomes. Many SAM domains have been shown to form helical polymer structures suggesting that SAM modules can be used to create large protein complexes in the cell. Because many polymeric SAM domains form heterogenous and insoluble aggregates that are experimentally intractable when isolated, it is likely that many polymeric SAM domains have gone uncharacterized. We, therefore, developed a method to maintain polymeric SAM domains in a soluble form that allowed rapid screening for potential SAM polymers. SAM domains were expressed as fusions to a super-negatively charged green fluorescent protein (negGFP). The negGFP imparts three useful properties to the SAM domains: (1) the charge helps to maintain solubility; (2) the charge leads to reliable migration toward the cathode on native gels; and (3) the fluorescence emission allows visualization in crude extracts. Using the negGFP-SAM fusions, we screened a large library of human SAM domains for polymerization using a native gel screen. A selected set of hSAM domains were then purified and examined for true polymer formation by electron microscopy. In this manner, we identified a set of new potential SAM polymers: ANKS3, Atherin, BicaudalC1, Caskin1, Caskin2, Kazrin, L3MBTL3, L3MBTL4, LBP, LiprinB1, LiprinB2, SAMD8, SAMD9, and STIM2. While further characterization will be necessary to verify that the SAM domains identified here truly form polymers, our results provide a much stronger working hypothesis for a large number of proteins that was possible from sequence analysis alone.

The molecular basis of the Caskin1 and Mint1 interaction with CASK.

Calcium/calmodulin-dependent serine protein kinase (CASK) is a conserved multi-domain scaffolding protein involved in brain development, synapse formation, and establishment of cell polarity. To accomplish these diverse functions, CASK participates in numerous protein-protein interactions. In particular, CASK forms competing CASK/Mint1/Velis and CASK/Caskin1/Velis tripartite complexes that physically associate with the cytoplasmic tail of neurexin, a transmembrane protein enriched at presynaptic sites. This study shows that a short linear EEIWVLRK peptide motif from Caskin1 is necessary and sufficient for binding CASK. We also identified the conserved binding site for the peptide on the CASK calmodulin kinase domain. A related EPIWVMRQ peptide from Mint1 was also discovered to be sufficient for binding. Searching all human proteins for the Mint1/Caskin1 consensus peptide ExIWVxR revealed that T-cell lymphoma invasion and metastasis 1 (TIAM1) contains a conserved EEVIWVRRE peptide that was also found to be sufficient for CASK binding in vitro. TIAM1 is well known for its role in tumor metastasis, but it also possesses overlapping cellular and neurological functions with CASK, suggesting a previously unknown cooperation between the two proteins. This new peptide interaction motif also explains how Caskin1 and Mint1 form competing complexes and suggests a new role for the cellular hub protein CASK.

Zinc binding drives sheet formation by the SAM domain of diacylglycerol kinase δ.

The diacylglycerol kinase (DGK) family of enzymes plays critical roles in lipid signaling pathways by converting diacylglycerol to phosphatidic acid, thereby downregulating signaling by the former and upregulating signaling by the latter second messenger. Ten DGK family isozymes have been identified to date, which possess different interaction motifs imparting distinct temporal and spatial control of DGK activity to each isozyme. Two DGK family members, δ and η, contain a sterile alpha motif (SAM) domain. The SAM domain of DGKδ1 forms helical polymers that are important for retaining the enzyme in cytoplasmic puncta, thereby inhibiting activity at the plasma membrane until pathway activation. Because zinc was found to be important for stabilizing the similar SAM polymers of the scaffolding protein Shank-3, we investigated the potential role of zinc in DGKδ SAM domain (DGKδSAM) assembly. We find that DGKδSAM binds zinc at multiple sites, driving the organization of the DGKδSAM into large sheets of polymers. Moreover, a mutant DGKδ containing a SAM domain refractory to zinc binding diminishes the formation of cytoplasmic puncta, shows partially impaired regulation of transport to the plasma membrane, and lacks the ability to inhibit the formation of CopII coated vesicles. These results suggest that zinc may play an important role in the assembly and physiology of the DGKδ isozyme.

Regulation of enzyme localization by polymerization: polymer formation by the SAM domain of diacylglycerol kinase delta1.

The diacylglycerol kinase (DGK) enzymes function as regulators of intracellular signaling by altering the levels of the second messengers, diacylglycerol and phosphatidic acid. The DGK delta and eta isozymes possess a common protein-protein interaction module known as a sterile alpha-motif (SAM) domain. In DGK delta, SAM domain self-association inhibits the translocation of DGK delta to the plasma membrane. Here we show that DGK delta SAM forms a polymer and map the polymeric interface by a genetic selection for soluble mutants. A crystal structure reveals that DGKSAM forms helical polymers through a head-to-tail interaction similar to other SAM domain polymers. Disrupting polymerization by polymer interface mutations constitutively localizes DGK delta to the plasma membrane. Thus, polymerization of DGK delta regulates the activity of the enzyme by sequestering DGK delta in an inactive cellular location. Regulation by dynamic polymerization is an emerging theme in signal transduction.