Generation and characterization of Ccdc28b mutant mice links the Bardet-Biedl associated gene with mild social behavioral phenotypes.

CCDC28B (coiled-coil domain-containing protein 28B) was identified as a modifier in the ciliopathy Bardet-Biedl syndrome (BBS). Our previous work in cells and zebrafish showed that CCDC28B plays a role regulating cilia length in a mechanism that is not completely understood. Here we report the generation of a Ccdc28b mutant mouse using CRISPR/Cas9 (Ccdc28b mut). Depletion of CCDC28B resulted in a mild phenotype. Ccdc28b mut animals i) do not present clear structural cilia affectation, although we did observe mild defects in cilia density and cilia length in some tissues, ii) reproduce normally, and iii) do not develop retinal degeneration or obesity, two hallmark features of reported BBS murine models. In contrast, Ccdc28b mut mice did show clear social interaction defects as well as stereotypical behaviors. This finding is indeed relevant regarding CCDC28B as a modifier of BBS since behavioral phenotypes have been documented in BBS. Overall, this work reports a novel mouse model that will be key to continue evaluating genetic interactions in BBS, deciphering the contribution of CCDC28B to modulate the presentation of BBS phenotypes. In addition, our data underscores a novel link between CCDC28B and behavioral defects, providing a novel opportunity to further our understanding of the genetic, cellular, and molecular basis of these complex phenotypes.

A novel form of Deleted in breast cancer 1 (DBC1) lacking the N-terminal domain does not bind SIRT1 and is dynamically regulated in vivo.

The protein Deleted in Breast Cancer-1 is a regulator of several transcription factors and epigenetic regulators, including HDAC3, Rev-erb-alpha, PARP1 and SIRT1. It is well known that DBC1 regulates its targets, including SIRT1, by protein-protein interaction. However, little is known about how DBC1 biological activity is regulated. In this work, we show that in quiescent cells DBC1 is proteolytically cleaved, producing a protein (DN-DBC1) that misses the S1-like domain and no longer binds to SIRT1. DN-DBC1 is also found in vivo in mouse and human tissues. Interestingly, DN-DBC1 is cleared once quiescent cells re-enter to the cell cycle. Using a model of liver regeneration after partial hepatectomy, we found that DN-DBC1 is down-regulated in vivo during regeneration. In fact, WT mice show a decrease in SIRT1 activity during liver regeneration, coincidentally with DN-DBC1 downregulation and the appearance of full length DBC1. This effect on SIRT1 activity was not observed in DBC1 KO mice. Finally, we found that DBC1 KO mice have altered cell cycle progression and liver regeneration after partial hepatectomy, suggesting that DBC1/DN-DBC1 transitions play a role in normal cell cycle progression in vivo after cells leave quiescence. We propose that quiescent cells express DN-DBC1, which either replaces or coexist with the full-length protein, and that restoring of DBC1 is required for normal cell cycle progression in vitro and in vivo. Our results describe for the first time in vivo a naturally occurring form of DBC1, which does not bind SIRT1 and is dynamically regulated, thus contributing to redefine the knowledge about its function.

BBS4 regulates the expression and secretion of FSTL1, a protein that participates in ciliogenesis and the differentiation of 3T3-L1.

Bardet-Biedl syndrome is a model ciliopathy. Although the characterization of BBS proteins has evidenced their involvement in cilia, extraciliary functions for some of these proteins are also being recognized. Importantly, understanding both cilia and cilia-independent functions of the BBS proteins is key to fully dissect the cellular basis of the syndrome. Here we characterize a functional interaction between BBS4 and the secreted protein FSTL1, a protein linked to adipogenesis and inflammation among other functions. We show that BBS4 and cilia regulate FSTL1 mRNA levels, but BBS4 also modulates FSTL1 secretion. Moreover, we show that FSTL1 is a novel regulator of ciliogenesis thus underscoring a regulatory loop between FSTL1 and cilia. Finally, our data indicate that BBS4, cilia and FSTL1 are coordinated during the differentiation of 3T3-L1 cells and that FSTL1 plays a role in this process, at least in part, by modulating ciliogenesis. Therefore, our findings are relevant to fully understand the development of BBS-associated phenotypes such as obesity.

Basal body proteins regulate Notch signaling through endosomal trafficking.

Proteins associated with primary cilia and basal bodies mediate numerous signaling pathways, but little is known about their role in Notch signaling. Here, we report that loss of the Bardet-Biedl syndrome proteins BBS1 or BBS4 produces increased Notch-directed transcription in a zebrafish reporter line and in human cell lines. Pathway overactivation is accompanied by reduced localization of Notch receptor at both the plasma membrane and the cilium. In Drosophila mutants, overactivation of Notch can result from receptor accumulation in endosomes, and recent studies implicate ciliary proteins in endosomal trafficking, suggesting a possible mechanism by which overactivation occurs in BBS mutants. Consistent with this, we observe genetic interaction of BBS1 and BBS4 with the endosomal sorting complexes required for transport (ESCRT) gene TSG101 and accumulation of receptor in late endosomes, reduced endosomal recycling and reduced receptor degradation in lysosomes. We observe similar defects with disruption of BBS3. Loss of another basal body protein, ALMS1, also enhances Notch activation and the accumulation of receptor in late endosomes, but does not disrupt recycling. These findings suggest a role for these proteins in the regulation of Notch through endosomal trafficking of the receptor.

Regulation of ack-family nonreceptor tyrosine kinases.

Ack family non-receptor tyrosine kinases are unique with regard to their domain composition and regulatory properties. Human Ack1 (activated Cdc42-associated kinase) is ubiquitously expressed and is activated by signals that include growth factors and integrin-mediated cell adhesion. Stimulation leads to Ack1 autophosphorylation and to phosphorylation of additional residues in the C-terminus. The N-terminal SAM domain is required for full activation. Ack1 exerts some of its effects via protein-protein interactions that are independent of its kinase activity. In the basal state, Ack1 activity is suppressed by an intramolecular interaction between the catalytic domain and the C-terminal region. Inappropriate Ack1 activation and signaling has been implicated in the development, progression, and metastasis of several forms of cancer. Thus, there is increasing interest in Ack1 as a drug target, and studies of the regulatory properties of the enzyme may reveal features that can be exploited in inhibitor design.

PTB domain-directed substrate targeting in a tyrosine kinase from the unicellular choanoflagellate Monosiga brevicollis.

Choanoflagellates are considered to be the closest living unicellular relatives of metazoans. The genome of the choanoflagellate Monosiga brevicollis contains a surprisingly high number and diversity of tyrosine kinases, tyrosine phosphatases, and phosphotyrosine-binding domains. Many of the tyrosine kinases possess combinations of domains that have not been observed in any multicellular organism. The role of these protein interaction domains in M. brevicollis kinase signaling is not clear. Here, we have carried out a biochemical characterization of Monosiga HMTK1, a protein containing a putative PTB domain linked to a tyrosine kinase catalytic domain. We cloned, expressed, and purified HMTK1, and we demonstrated that it possesses tyrosine kinase activity. We used immobilized peptide arrays to define a preferred ligand for the third PTB domain of HMTK1. Peptide sequences containing this ligand sequence are phosphorylated efficiently by recombinant HMTK1, suggesting that the PTB domain of HMTK1 has a role in substrate recognition analogous to the SH2 and SH3 domains of mammalian Src family kinases. We suggest that the substrate recruitment function of the noncatalytic domains of tyrosine kinases arose before their roles in autoinhibition.

Regulation of Ack1 localization and activity by the amino-terminal SAM domain.

BACKGROUND: The mechanisms that regulate the activity of the nonreceptor tyrosine kinase Ack1 (activated Cdc42-associated kinase) are poorly understood. The amino-terminal region of Ack1 is predicted to contain a sterile alpha motif (SAM) domain. SAM domains share a common fold and mediate protein-protein interactions in a wide variety of proteins. Here, we addressed the importance of the Ack1 SAM domain in kinase activity. RESULTS: We used immunofluorescence and Western blotting to show that Ack1 deletion mutants lacking the N-terminus displayed significantly reduced autophosphorylation in cells. A minimal construct comprising the N-terminus and kinase domain (NKD) was autophosphorylated, while the kinase domain alone (KD) was not. When expressed in mammalian cells, NKD localized to the plasma membrane, while KD showed a more diffuse cytosolic localization. Co-immunoprecipitation experiments showed a stronger interaction between full length Ack1 and NKD than between full length Ack1 and KD, indicating that the N-terminus was important for Ack1 dimerization. Increasing the local concentration of purified Ack1 kinase domain at the surface of lipid vesicles stimulated autophosphorylation and catalytic activity, consistent with a requirement for dimerization and trans-phosphorylation for activity. CONCLUSIONS: Collectively, the data suggest that the N-terminus of Ack1 promotes membrane localization and dimerization to allow for autophosphorylation.

Cancer-associated mutations activate the nonreceptor tyrosine kinase Ack1.

Ack1 is a nonreceptor tyrosine kinase that participates in tumorigenesis, cell survival, and migration. Relatively little is known about the mechanisms that regulate Ack1 activity. Recently, four somatic missense mutations of Ack1 were identified in cancer tissue samples, but the effects on Ack1 activity, and function have not been described. These mutations occur in the N-terminal region, the C-lobe of the kinase domain, and the SH3 domain. Here, we show that the cancer-associated mutations increase Ack1 autophosphorylation in mammalian cells without affecting localization and increase Ack1 activity in immune complex kinase assays. The cancer-associated mutations potentiate the ability of Ack1 to promote proliferation and migration, suggesting that point mutation is a mechanism for Ack1 deregulation. We propose that the C-terminal Mig6 homology region (MHR) (residues 802-990) participates in inhibitory intramolecular interactions. The isolated kinase domain of Ack1 interacts directly with the MHR, and the cancer-associated E346K mutation prevents binding. Likewise, mutation of a key hydrophobic residue in the MHR (Phe(820)) prevents the MHR-kinase interaction, activates Ack1, and increases cell migration. Thus, the cancer-associated mutation E346K appears to destabilize an autoinhibited conformation of Ack1, leading to constitutively high Ack1 activity.