The crystal structure of the Physarum polycephalum actin-fragmin kinase: an atypical protein kinase with a specialized substrate-binding domain

Steinbacher, Stefan; Hof, Peter; Eichinger, Ludwig M.; Schleicher, Michael; Gettemans, Jan; Vandekerckhove, Joël; Huber, Robert; Benz, Jörg

doi:10.1093/emboj/18.11.2923

Cited by 35 publications

(44 citation statements)

References 30 publications

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“…This unique architecture likely restrains ceFam20 from undergoing highly dynamic conformational changes. To the best of our knowledge, the sole active protein kinase with a hydrophilic residue (Ser) at the Phe position in the DFG motif is AFK from slime mold (42). Of note, the αC in AFK is situated at a similar position as the α6 in ceFam20 (Fig.…”

Section: Resultsmentioning

confidence: 96%

Crystal structure of the Golgi casein kinase

Xiao

Tagliabracci

Wen

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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The family with sequence similarity 20 (Fam20) kinases phosphorylate extracellular substrates and play important roles in biomineralization. Fam20C is the Golgi casein kinase that phosphorylates secretory pathway proteins within Ser-x-Glu/pSer motifs. Mutations in Fam20C cause Raine syndrome, an osteosclerotic bone dysplasia. Here we report the crystal structure of the Fam20C ortholog from Caenorhabditis elegans. The nucleotide-free and Mn/ ADP-bound structures unveil an atypical protein kinase-like fold and highlight residues critical for activity. The position of the regulatory αC helix and the lack of an activation loop indicate an architecture primed for efficient catalysis. Furthermore, several distinct elements, including the presence of disulfide bonds, suggest that the Fam20 family diverged early in the evolution of the protein kinase superfamily. Our results reinforce the structural diversity of protein kinases and have important implications for patients with disorders of biomineralization.P rotein phosphorylation is a fundamental mechanism that regulates numerous physiological processes (1, 2). Protein kinases have evolved to function as dynamic switches relaying signals in response to various stimuli by transferring a phosphate from ATP to target proteins (3-5). Most phosphoproteins are found within the cell, residing in the nuclei and cytosol; however, secreted proteins, including casein and other members of the secretory calciumbinding phosphoprotein family, are phosphorylated as well (6). We recently identified a family of kinases that reside in the secretory pathway and function to phosphorylate extracellular substrates (7,8). One member of this family, Fam20C (family with sequence similarity 20, member C), is the physiological casein kinase that phosphorylates multiple secreted proteins within a Ser-x-Glu/pSer motif (7, 9, 10). The importance of this discovery is underscored by the fact that some 75% of the phosphoproteins identified in human serum and cerebrospinal fluid contain phosphate within this motif (11-13). Furthermore, mutations in FAM20C cause Raine syndrome, a deadly osteosclerotic bone dysplasia characterized by generalized osteosclerosis, ectopic calcifications, and characteristic facial features (14-16). Most individuals with Raine syndrome die within a few weeks after birth; however, nonlethal cases with dental abnormalities and clinical features of hypophosphatemia have been reported (17,18). Loss of Fam20C in mice also results in severe bone and tooth anomalies, as well as hypophosphatemia (19)(20)(21).Two other closely related Fam20C paralogs, Fam20A and Fam20B, are present in humans (22). Fam20B is ubiquitously expressed and phosphorylates xylose within the tetrasaccharide linkage region of proteoglycans (23). This phosphorylation event may influence glycosaminoglycan biosynthesis (24). Genetic deletion of Fam20B in mice results in embryonic lethality at E13.5, and mutations in Danio rerio result in reduced cartilage matrix production and skeletal defects (19,25). The substra...

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Section: Resultsmentioning

confidence: 96%

Crystal structure of the Golgi casein kinase

Xiao

Tagliabracci

Wen

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…S1). However, the strain was not detected in distantly related APKs, such as actin-fragmin kinase (29).…”

Section: Catalytic Loop Strain Is Conserved In Epks and Elks That Arementioning

confidence: 91%

Identification of a hidden strain switch provides clues to an ancient structural mechanism in protein kinases

Oruganty¹,

Talathi²,

Wood³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The protein kinase catalytic domain contains several conserved residues of unknown functions. Here, using a combination of computational and experimental approaches, we show that the function of some of these residues is to maintain the backbone geometry of the active site in a strained conformation. Specifically, we find that the backbone geometry of the catalytically important HRD motif deviates from ideality in high-resolution structures and the strained geometry results in favorable hydrogen bonds with conserved noncatalytic residues in the active site. In particular, a conserved aspartate in the F-helix hydrogen bonds to the strained HRD backbone in diverse eukaryotic and eukaryotic-like protein kinase crystal structures. Mutations that alter this hydrogen-bonding interaction impair catalytic activity in Aurora kinase. Although the backbone strain is present in most active conformations, several inactive conformations lack the strain because of a peptide flip in the HRD backbone. The peptide flip is correlated with loss of hydrogen bonds with the F-helix aspartate as well as with other interactions associated with kinase regulation. Within protein kinases that are regulated by activation loop phosphorylation, the strained residue is an arginine, which coordinates with the activation loop phosphate. Based on analysis of strain across the protein kinase superfamily, we propose a model in which backbone strain co-evolved with conserved residues for allosteric control of catalytic activity. Our studies provide new clues for the design of allosteric protein kinase inhibitors.allostery | conformational strain | DFG flip | hydrophobic spine | Ramachandran plot

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“…11). We chose the Physarum kinase as a template because it is the most closely related kinase with a solved crystal structure (48) and the only known structure that shares the sequence pattern DXXXXNXDR with PI4KII (in motif 6, see Fig. 5).…”

Section: Fig 8 Sequence and Expression Of Full-length And Truncatedmentioning

confidence: 99%

Analysis of the Catalytic Domain of Phosphatidylinositol 4-Kinase Type II

Baryłko¹,

Włodarski

Binns³

et al. 2002

Journal of Biological Chemistry

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Phosphatidylinositol (PtdIns) 4-kinases catalyze the conversion of PtdIns to PtdIns 4-phosphate, the major precursor of phosphoinositides that regulates a vast array of cellular processes. Based on enzymatic differences, two classes of PtdIns 4-kinase have been distinguished termed Types II and III. Type III kinases, which belong to the phosphatidylinositol (PI) 3/4-kinase family, have been extensively characterized. In contrast, little is known about the Type II enzymes (PI4KIIs), which have been cloned and sequenced very recently. PI4KIIs bear essentially no sequence similarity to other protein or lipid kinases; hence, they represent a novel and distinct branch of the kinase superfamily. Here we define the minimal catalytic domain of a rat PI4KII isoform, PI4KII␣, and identify conserved amino acid residues required for catalysis. We further show that the catalytic domain by itself determines targeting of the kinase to membrane rafts. To verify that the PI4KII family extends beyond mammalian sources, we expressed and characterized Drosophila PI4KII and its catalytic domain. Depletion of PI4KII from Drosophila cells resulted in a severe reduction of PtdIns 4-kinase activity, suggesting the in vivo importance of this enzyme.Phosphoinositides (PIs), 1 especially PtdIns 4,5-biphosphate and PtdIns 3,4,5-trisphosphate, are key regulators of many cell functions (1-5). The major, so-called canonical pathway leading to the synthesis of these two PIs begins with phosphorylation of PtdIns on the D-4 hydroxyl by PtdIns 4-kinases. Two classes of PtdIns 4-kinase, Types II and III, have been identified (6). They are distinguished primarily by differences in their enzymatic properties; Type II kinases have 3-7-fold lower K m values for PtdIns and ATP than Type III kinases, they are about 20-fold more sensitive than Type III kinases to inhibition by adenosine, and they are insensitive to inhibition by wortmannin, a drug that inhibits the Type III kinases at submicromolar concentrations (for reviews, see Refs. 7 and 8). In addition, a monoclonal antibody (4C5G) raised against partially purified PI4KII from bovine brain selectively inhibits Type II kinase activities (9).In 1993 Flanagan et al. (10) reported the first sequence of a PtdIns 4-kinase, a 125-kDa enzyme purified a year earlier from Saccharomyces cerevisiae (11). The enzymatic properties of this yeast kinase, now known as Pik1p, placed it in the Type III class. Shortly thereafter, a 216-kDa Type III kinase, Stt4p, was also cloned from yeast (12). Mammalian orthologs of Stt4p and Pik1p, sometimes designated PI4KIII␣ and PI4KIII␤, respectively, were soon cloned and sequenced (13-18). The two forms of PI4KIII contain two regions of homology, the C-terminal catalytic domain and an internal "lipid kinase unique" domain. Homologous domains are also found in PI 3-kinases and in a group of related Ser/Thr protein kinases, including RAFT, TOR, ATM, and DNA-dependent protein kinase (19). Therefore, Type III PtdIns 4-kinase have been classified as members of the PI3/PI4-kinase fa...

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The crystal structure of the Physarum polycephalum actin-fragmin kinase: an atypical protein kinase with a specialized substrate-binding domain

Cited by 35 publications

References 30 publications

Crystal structure of the Golgi casein kinase

Crystal structure of the Golgi casein kinase

Identification of a hidden strain switch provides clues to an ancient structural mechanism in protein kinases

Analysis of the Catalytic Domain of Phosphatidylinositol 4-Kinase Type II

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