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The Fam20 proteins are novel kinases that phosphorylate secreted proteins and proteoglycans. Fam20C phosphorylates hundreds of secreted proteins and is activated by the pseudokinase Fam20A. Fam20B phosphorylates a xylose residue to regulate proteoglycan synthesis. Despite these wide-ranging and important functions, the molecular and structural basis for the regulation and substrate specificity of these kinases are unknown. Here we report molecular characterizations of all three Fam20 kinases, and show that Fam20C is activated by the formation of an evolutionarily conserved homodimer or heterodimer with Fam20A. Fam20B has a unique active site for recognizing Galβ1-4Xylβ1, the initiator disaccharide within the tetrasaccharide linker region of proteoglycans. We further show that in animals the monomeric Fam20B preceded the appearance of the dimeric Fam20C, and the dimerization trait of Fam20C emerged concomitantly with a change in substrate specificity. Our results provide comprehensive structural, biochemical, and evolutionary insights into the function of the Fam20 kinases., Fam20 proteins are kinases that phosphorylate secreted proteins and proteoglycans. Here the authors present the crystal structures of the three Fam20 kinases and their complexes and give mechanistic insights into substrate recognition and the function and regulation of this kinase family.

SummaryThe existence of extracellular phosphoproteins has been acknowledged for over a century. However, research in this area has been undeveloped largely because the kinases that phosphorylate secreted proteins have escaped identification. Fam20C is a kinase that phosphorylates S-x-E/pS motifs on proteins in milk and in the extracellular matrix of bones and teeth. Here, we show that Fam20C generates the majority of the extracellular phosphoproteome. Using CRISPR/Cas9 genome editing, mass spectrometry, and biochemistry, we identify more than 100 secreted phosphoproteins as genuine Fam20C substrates. Further, we show that Fam20C exhibits broader substrate specificity than previously appreciated. Functional annotations of Fam20C substrates suggest roles for the kinase beyond biomineralization, including lipid homeostasis, wound healing, and cell migration and adhesion. Our results establish Fam20C as the major secretory pathway protein kinase and serve as a foundation for new areas of investigation into the role of secreted protein phosphorylation in human biology and disease.

Mutations in FAM20A cause tooth enamel defects known as Amelogenesis Imperfecta (AI) and renal calcification. We previously showed that Fam20A is a secretory pathway pseudokinase and allosterically activates the physiological casein kinase Fam20C to phosphorylate secreted proteins important for biomineralization (Cui et al., 2015). Here we report the nucleotide-free and ATP-bound structures of Fam20A. Fam20A exhibits a distinct disulfide bond pattern mediated by a unique insertion region. Loss of this insertion due to abnormal mRNA splicing interferes with the structure and function of Fam20A, resulting in AI. Fam20A binds ATP in the absence of divalent cations, and strikingly, ATP is bound in an inverted orientation compared to other kinases. Fam20A forms a dimer in the crystal, and residues in the dimer interface are critical for Fam20C activation. Together, these results provide structural insights into the function of Fam20A and shed light on the mechanism by which Fam20A mutations cause disease. DOI: http://dx.doi.org/10.7554/eLife.23990.001

Most eukaryotic cells elaborate several proteoglycans critical for transmitting biochemical signals into and between cells. However, the regulation of proteoglycan biosynthesis is not completely understood. We show that the atypical secretory kinase family with sequence similarity 20, member B (Fam20B) phosphorylates the initiating xylose residue in the proteoglycan tetrasaccharide linkage region, and that this event functions as a molecular switch to regulate subsequent glycosaminoglycan assembly. Proteoglycans from FAM20B knockout cells contain a truncated tetrasaccharide linkage region consisting of a disaccharide capped with sialic acid (Siaα2-3Galβ1-4Xylβ1) that cannot be further elongated. We also show that the activity of galactosyl transferase II (GalT-II, B3GalT6), a key enzyme in the biosynthesis of the tetrasaccharide linkage region, is dramatically increased by Fam20B-dependent xylose phosphorylation. Inactivating mutations in the GALT-II gene (B3GALT6) associated with Ehlers-Danlos syndrome cause proteoglycan maturation defects similar to FAM20B deletion. Collectively, our findings suggest that GalT-II function is impaired by loss of Fam20B-dependent xylose phosphorylation and reveal a previously unappreciated mechanism for regulation of proteoglycan biosynthesis.

Targeting eukaryotic proteins for deamidation modification is increasingly appreciated as a general bacterial virulence mechanism. Here, we present an atomic view of how a bacterial deamidase effector, cycle-inhibiting factor homolog in Burkholderia pseudomallei (CHBP), recognizes its host targets, ubiquitin (Ub) and Ub-like neural precursor cell expressed, developmentally down-regulated 8 (NEDD8), and catalyzes site-specific deamidation. Crystal structures of CHBP–Ub/NEDD8 complexes show that Ub and NEDD8 are similarly cradled by a large cleft in CHBP with four contacting surfaces. The pattern of Ub/NEDD8 recognition by CHBP resembles that by the E1 activation enzyme, which critically involves the Lys-11 surface in Ub/NEDD8. Close examination of the papain-like catalytic center reveals structural determinants of CHBP being an obligate glutamine deamidase. Molecular-dynamics simulation identifies Gln-31/Glu-31 of Ub/NEDD8 as one key determinant of CHBP substrate preference for NEDD8. Inspired by the idea of using the unique bacterial activity as a tool, we further discover that CHBP-catalyzed NEDD8 deamidation triggers macrophage-specific apoptosis, which predicts a previously unknown macrophage-specific proapoptotic signal that is negatively regulated by neddylation-mediated protein ubiquitination/degradation.

Protein phosphorylation is a fundamental mechanism regulating nearly every aspect of life. Several secreted proteins are phosphorylated, but the kinases responsible are unknown. We identified a family of atypical protein kinases which have N-terminal signal sequence and localize to the lumen of the endoplasmic reticulum and the Golgi apparatus. One of these kinases known as Fam20C is the Golgi casein kinase and it phosphorylates secretory pathway proteins within S-x-E motifs. Fam20C phosphorylates the caseins and many other secretory proteins including the small integrin-binding ligand, N-linked glycoproteins (SIBLINGs) implicated in biomineralization. Consequently, mutations in Fam20C cause an osteosclerotic bone dysplasia in humans known as Raine syndrome. Another family member, Fam20B, is a xylose kinase important in the maturation of proteoglycans. The function of other FAM 20C family members will also be addressed.

Although numerous extracellular phosphoproteins have been identified, the protein kinases within the secretory pathway have only recently been discovered, and their regulation is virtually unexplored. Fam20C is the physiological Golgi casein kinase, which phosphorylates many secreted proteins and is critical for proper biomineralization. Fam20A, a Fam20C paralog, is essential for enamel formation, but the biochemical function of Fam20A is unknown. Here we show that Fam20A potentiates Fam20C kinase activity and promotes the phosphorylation of enamel matrix proteins in vitro and in cells. Mechanistically, Fam20A is a pseudokinase that forms a functional complex with Fam20C, and this complex enhances extracellular protein phosphorylation within the secretory pathway. Our findings shed light on the molecular mechanism by which Fam20C and Fam20A collaborate to control enamel formation, and provide the first insight into the regulation of secretory pathway phosphorylation. DOI: http://dx.doi.org/10.7554/eLife.06120.001, eLife digest Some proteins must be modified in order to work effectively. One common modification is to add a phosphate group to the protein, which is performed by enzymes called protein kinases. Although most of the protein kinases work on proteins inside the cell, it was discovered recently that a small group of kinases work within the ‘secretory pathway’ and modify proteins that are released (or secreted) out of cells. One such secretory pathway kinase—called Fam20C—phosphorylates a wide range of secreted proteins and helps to ensure the proper development of bones and teeth. Specifically, Fam20C and a closely related protein called Fam20A are important for forming enamel, the hardest substance in human body, which makes up the outer surface of teeth. However, the exact role of Fam20A is unknown. Cui et al. now show that Fam20A binds to Fam20C, and this increases the ability of Fam20C to phosphorylate the proteins that form the ‘matrix’ that guides the deposition of the enamel minerals. Furthermore, mutations in Fam20A lead to the inefficient phosphorylation of enamel matrix proteins by Fam20C, and prevent proper enamel formation. The results raise the possibility that similar mechanisms of secretory kinase activation may also be important in other biological processes where many secreted proteins need to be phosphorylated rapidly. DOI: http://dx.doi.org/10.7554/eLife.06120.002

To understand the regulatory mechanisms governing unisexual flower development in cucumber, we conducted a systematic morphogenetic analysis of male and female flower development, examined the dynamic changes in expression of the C-class floral organ identity gene CUM1, and assessed the extent of DNA damage in inappropriate carpels of male flowers. Accordingly, based on the occurrence of distinct morphological events, we divided the floral development into 12 stages ranging from floral meristem initiation to anthesis. As a result of our investigation we found that the arrest of stamen development in female flowers, which occurs just after the differentiation between the anther and filament, is mainly restricted to the primordial anther, and that it is coincident with down-regulation of CUM1 gene expression. In contrast, the arrest of carpel development in the male flowers occurs prior to the differentiation between the stigma and ovary, given that no indication of ovary differentiation was observed even though CUM1 gene expression remained detectable throughout the development of the stigma-like structures. Although the male and female reproductive organs have distinctive characteristics in terms of organ differentiation, there are two common features regarding organ arrest. The first is that the arrest of the inappropriate organ does not affect the entirety of the organ uniformly but occurs only in portions of the organs. The second feature is that all the arrested portions in both reproductive organs are spore-bearing parts.

Bacterial virulence often relies on secreted effectors that modulate eukaryotic signal transduction. Recent studies provide a collection of examples in which bacterial effectors carry out unprecedented posttranslational modifications of key signaling molecules or organize a new signaling network. OspF and YopJ families of effectors use novel modification activities to block kinase phosphoactivation. Targeting of the ubiquitin system by IpaH and Cif/CHBP families provides insights into host ubiquitin signaling. Manipulation of small GTPases by VopS/IbpA and SidM suggests previously underappreciated regulation of signaling. Several other effectors, including SifA and EspG, organize newly discovered signaling networks in membrane trafficking. Studies of these effectors can generate new knowledge in enzyme catalysis and provide new angles for furthering our understanding of biochemical regulation of important signaling pathways.

Gee-Up, NEDD8 Diverse bacterial pathogens, such as Burkholderia pseudomallei and enteropathogenic Escherichia coli (EPEC), inject effector proteins into eukaryotic host cells to subvert host cell biology. Cui et al. (p. 1215 ; published online 5 August) show that certain bacterial effector proteins promoted the glutamine deamidation of ubiquitin and the ubiquitin-related protein, NEDD8. The modification of ubiquitin inhibited the formation of polyubiquitin chains while, unexpectedly, the same chemical alteration on NEDD8 interfered with the cell cycle, offering insights not only into the cytopathic effects of bacterial infection but also into mechanisms that may operate during viral infection and cancer.

Pathogenic bacteria deliver effector proteins into host cells through the type III secretion apparatus to modulate the host function. We identify a family of proteins, homologous to the type III effector Cif from enteropathogenic Escherichia coli , in pathogens including Yersinia , Photorhabdus , and Burkholderia that contain functional type III secretion systems. Like Cif, this family of proteins is capable of arresting the host cell cycle at G 2 /M. Structure of one of the family members, Cif homolog in Burkholderia pseudomallei (CHBP), reveals a papain-like fold and a conserved Cys-His-Gln catalytic triad despite the lack of primary sequence identity. For CHBP and Cif, only the putative catalytic Cys is susceptible to covalent modification by E-64, a specific inhibitor of papain-like cysteine proteases. Unlike papain-like enzymes where the S2 site is the major determinant of cleavage-site specificity, CHBP has a characteristic negatively charged pocket occupying surface areas corresponding to the S1/S1′ site in papain-like proteases. The negative charge is provided by a conserved aspartate, and the pocket best fits an arginine, as revealed by molecular docking analysis. Mutation analysis establishes the essential role of the catalytic triad and the negatively charged pocket in inducing cell cycle arrest in host cells. Our results demonstrate that bacterial pathogens have evolved a unique papain-like hydrolytic activity to block the normal host cell cycle progression.