Abstract
The previous decades have seen an explosion in our understanding of protein kinase function in human health and disease. Hundreds of unique kinase structures have been solved, allowing us to create generalized rules for catalysis, assign roles for communities within the catalytic core, and develop specific drugs for targeting various pathways. While our understanding of intracellular kinases has developed at a fast rate, our exploration into extracellular kinases has just begun. In this review we will cover the secreted protein kinase families found in humans, bacteria, and parasites.
The extracellular space is fundamentally different than the cytoplasm of a cell. It is primarily an oxidizing environment with a drastically different composition of ions and small molecules 1. Secreted proteins are often rich in cysteines, which can form disulfide bonds to stabilize the protein. In contrast, cytosolic proteins rarely contain disulfides in the reducing milieu of the intracellular space due to high levels of glutathione and cellular reducing enzymes 2, 3. Secreted proteins often play specialized roles to coordinate events as diverse as hormone signaling, wound repair, extracellular matrix remodeling, and pathogenicity 4, 5.
In eukaryotes, proteins destined for secretion typically travel through the secretory pathway comprising the endoplasmic reticulum (ER) and Golgi apparatus. These compartments are isolated from the cytoplasm and more closely resemble the extracellular environment 1, 6. Proteins can enter the lumen of the ER either co-translationally or post-translationally (Figure 1A). Co-translationally translocated proteins are identified by the signal-recognition particle (SRP), which binds to short N-terminally encoded signal peptides or transmembrane helices. The SRP halts translation and brings the nascent mRNA chain to the SRP receptor located on the ER. For post-translationally translocated proteins, cytosolic chaperones such as Hsp70 bind to hydrophobic patches and stabilize the polypeptide chain in an unfolded and translocation competent state before it is brought to the Sec63 complex. Sec61 forms an aqueous channel and works in conjunction with both the SRP receptor and the Sec63 complex to feed the polypeptide chains into the lumen of the ER 7.
Secreted Human Kinases
Extracellular and secretory pathway phosphorylation in humans is involved in numerous cellular and physiological processes such as bone mineralization, hormone signaling, extracellular matrix remodeling, and embryogenesis, among others 4, 8–12. Disruption of the extracellular phosphorylation landscape has dramatic effects and can results in diseases such as osteosclerotic bone dysplasia, hypophosphatemia, and gingival hyperplasia 13–15 (Table 1). While the importance of proper extracellular phosphorylation is widely accepted, the enzymes responsible for these events have eluded identification and confounded attempts to study detailed mechanisms behind these diseases.
Table 1:
Activity | Substrates Known | Associated Diseases | |
---|---|---|---|
Fam20A | No | -- | Amelogenesis impefecta. Gingival hyperplasia. Enamel-renal syndrome |
Fam20B | Yes* | Yes | Chondrosarcoma, Periochondral bone defects. Ossification defects in long bones. Cartilidge defects |
Fam20C | Yes* | Yes | Raine Syndrome, FGF23-dependent hypophosphatemic rickets |
Fam69A | Unknown | -- | Schizophrenia, bipolar disorder, multiple sclerosis |
Fam69B | Unknown | -- | Autism spectrum disorders |
Fam69C | Unknown | -- | -- |
VLK | Yes* | Yes | Skeletal disorders. Defective differentiation of embryonic stem cells |
DIA1 | Yes | No | Autism |
DIA1R | Yes | No | Fragile X Syndrome, Kabuki Syndrome |
Fam198A | Unknown | -- | -- |
Fam198B | Unknown | -- | -- |
Asterisk denotes those shown experimentally to have kinase activity.
Interestingly, one of the earliest recorded instances of phosphorylation and, in retrospect, the existence of protein kinases, came from secreted milk and egg yolk proteins 16, 17. Following the discovery of phosphorylase kinase 18, 19, the protein kinase field exploded, culminating in the publication of the “Human Kinome” 20. However, the molecular identification of the kinases that phosphorylate extracellular proteins, including casein from breast milk, remained unknown. In fact, all the known human protein kinases localized to the cytosol or nucleus and therefore would be highly unlikely to encounter casein and other secreted proteins within the lumen of the secretory pathway or outside of the cell.
Elegant biochemical studies from the laboratory of Lorenzo Pinna had uncovered a casein kinase activity from highly enriched Golgi fractions from the lactating mammary gland 21. This enzyme, termed Golgi casein kinase (GCK) or Golgi-enriched fraction casein kinase (GEF-CK), preferred manganese over magnesium as the activating divalent cation and was insensitive to the broad spectrum protein kinase inhibitor, staurosporine 21. Importantly, the GCK specifically phosphorylated Ser residues within the consensus sequence Ser-x-Glu/pSer (where x is any amino acid), a motif commonly found to be phosphorylated in the caseins and a plethora of other secreted phosphoproteins 22, 23. Despite the extensive biochemical characterization, the molecular identify of the GCK remained unknown and was therefore annotated as an “orphan enzyme”.
In 2008, the Irvine lab discovered that the four jointed (fj) protein from Drosophila was a Golgi kinase that phosphorylates extracellular cadherin domains 24. In 2012, the Dixon lab undertook a bioinformatic approach using the human four jointed (Fjx1) as a query in a PSI-BLAST search and identified the Fam20 family of predicted human secretory pathway kinases (Figure 2A). Interestingly, these kinases were so different form canonical kinases that they were not found in the first iteration of the human kinome 20. The Fam20-proteins contain signal peptides that would be predicted to orient their catalytic domains facing the lumen of the secretory pathway or outside of the cell (Figure 1B). Among these members, Fam20C was found to be the physiological GCK 25. In addition to the Fam20-family, the Pawlowski laboratory bioinformatically identified another family of putative secretory pathway kinases related in sequence to Vertebrate Lonesome kinase (VLK) (Figure 2B), which was shown by the Whitman laboratory in 2014 to be a secreted tyrosine kinase 26.
Fam20A/B/C
The flagship member of the Fam20 family of kinases is Fam20C. Fam20C is the physiological Golgi casein kinase and more recently, has been identified as the phosvitin kinase, an abundant egg yolk protein that has been known to be highly phosphorylated since the beginning of the 20th century 17, 27. Fam20C is a secreted protein whose activity can be purified from whey extracts of milk. Quantitative phosphoproteomics from the conditioned medium of Fam20C knockout and wildtype HepG2 cell lines implicated Fam20C in the phosphorylation of nearly 70% of all secretory phosphoproteins 5. Hypomorphic mutations in the kinase domain are associated with a form of osteosclerotic bone dysplasia (Raine Syndrome) 28, as well as FGF23-dependent hypophosphatemia 29. The crystal structure of Fam20C revealed an atypical kinase fold consisting of numerous highly conserved cysteines as well as multiple helical and loop insertions that form a shell around the enzyme (Figure 2C). These extra secondary structural elements, along with a few missing kinase sequence motifs, likely confounded initial bioinformatic attempts to classify it as a protein kinase.
In addition to phosphorylating the small-integrin binding N-linked glycoproteins (SIBLING’s) to regulate biomineralization 25, 30, Fam20C also phosphorylates the phosphate regulating hormone FGF23 ultimately leading to elevated blood phosphate levels 15. Mice and humans with Fam20C inactivating mutations have hypophosphatemic rickets with elevated levels of biologically active FGF23 14, 29, 31. Fam20C also appears to fine-tune ER redox homeostasis by phosphorylating ER oxidoreductin protein 1 alpha (Ero1a) and increasing its oxidase activity and oxidative folding of ER proteins 32. Moreover, Fam20C phosphorylates the histidine-rich calcium-binding protein HRC to prevent cardiac arrhythmia 33. Interestingly, a common Ser96Ala genetic variant in HRC correlates with ventricular arrhythmias in humans with dilated cardiomyopathy 34. Cardiac specific deletion of Fam20C results in age and pressure overload induced heart failure by phosphorylation and alteration of the SR proteins calsequestrin-2 and STIM1, indicating a critical role for Fam20C in cardiac pathophysiology 35.
Fam20C also works in conjunction with Fam20A. Despite high sequence similarity between the two proteins, Fam20A possesses mutations in key catalytic residues that result in an inactive pseudokinase. Structural studies of Fam20A revealed unique disulfide bond patterns and ATP bound in an inverted orientation 36. Fam20A is associated with diseases such as amelogenesis imperfecta, gingival hyperplasia and enamel-renal syndrome 37–39. This, along with expression data showing coordinated expression patterns of Fam20A/C during mammary gland lactation, suggests an alternate role for the pseudokinase 40. Indeed, Fam20A forms a heterotetramer with Fam20C (Figure 2D) 41 and serves to increase Fam20C activity during periods of enhanced phosphoprotein secretion such as enamel formation and lactation. Fam20A appears to promote extracellular accumulation of Fam20C and modulate extracellular mineralization by regulating Fam20C localization 42. Because Fam20A is not expressed in all tissues that contain Fam20C, an alternate mechanism for Fam20C activation has recently emerged in which sphingosine activates Fam20C in vitro and in cells 43, 44. Interestingly Fam20C possesses remote sequence similarity to CotH, which regulates sporulation and germination in several spore-forming prokaryotic and eukaryotic pathogens 45.
The remaining Fam20 member, Fam20B, is a sugar kinase. Fam20B phosphorylates the xylose sugar within the tetrasaccharide linkage region of proteoglycans, leading to stimulation of galactosyltransferase II (GalT-II) activity and further extension of the polysaccharide chain 46, 47. Inactivating mutations of Fam20B in mouse models results in multiple anomalies associated with proper proteoglycan function. For example, conditional K14-Cre driven epithelial Fam20B knockout mice had supernumerary maxillary and mandibular incisors, with major defects in proteoglycan signaling 48. Fam20B was also identified in a zebrafish mutagenesis screen looking for defects in cartilage and perichondral bone. Mutations in Fam20B and Xylt1 resulted in precocious differentiation of perichondral osteoblasts and increased perichondral bone 49. In addition, Osr2 driven palate and kidney conditional knockouts resulted in chondrosarcoma and postnatal ossification defects in long bones 50. Intriguingly, Fam20B is the oldest member of the family, with homologs found as far back as Porifera (sea sponges) 41. Based on structural and sequence similarity it is hypothesized that Fam20C acquired key mutations that successfully converted Fam20B from a sugar kinase to a protein kinase 41 (Figure 2C and E).
VLK, Fam69 and DIA/DIAIR
In addition to the Fam20 family of secretory kinases, another family of tyrosine-kinase like proteins exists in the extracellular space. The first characterized member the tyrosine kinase-like branch of secretory pathway and extracellular kinases is the vertebrate lonesome kinase (VLK). VLK was initially cloned as a protein involved in differentiation of embryonic stem cells 51, 52. Intriguingly, VLK knockout mice die within the first day of life due to severe morphogenic defects in a wide variety of tissues 26. Homozygous loss-of-function variants in humans have recently been shown to be associated with skeletal disorders 53. These studies implicate VLK in proper organogenesis perhaps through embryonic stem cell differentiation. VLK is highly expressed in blood platelets where it is hypothesized to be involved in platelet activation and released upon degranulation to regulate wound healing, angiogenesis, and thrombosis. 26
Analysis of VLK overexpression and knockdown cells suggest that VLK is capable of phosphorylating multiple proteins within the secretory pathway and the extracellular space. Moreover, VLK phosphorylates physiologically relevant sites on metalloproteinase 1 (MMP1) and ERP29. Mass spectrometry analysis of p-Tyr-containing proteins in VLK expressing cells show a broad range of potential substrates including osteopontin, insulin growth factor binding proteins, and follistatin related protein-1 26.
DIA1 and DIA1R were initially implicated in Autism spectrum disorders and X-linked mental retardation 54. DIA1 is found as far back as cnidarians, while DIA1R is found exclusively in vertebrates 55. Both genes are ubiquitously expressed and are enriched in human brain tissue 54. Very little is known about these proteins outside of their potential link to neurological diseases. Analysis of cells overexpressing DIA1 show an increase in phosphosites with a preference for a [ST]P and Sx[DE]/Sxx[DE]/S[DE] motifs, 56.
Little is known about the Fam69 subfamily of secretory pathway kinases. Primary and secondary sequence analysis suggests similarity to VLK. Both Fam69 and DIA/DIAR family member proteins are predicted to localize to the secretory pathway; the former possessing type-II transmembrane domains which orient the kinase into the lumen of the ER, and the latter possessing signal peptides (Figure 1B). While their substrates and activities have yet to be characterized, all family members are implicated in neurological diseases including autism, fragile X syndrome (FXS), schizophrenia, and congenital mental retardation 57–59. Tissue expression analysis of these proteins reveal high expression of Fam69B/C in the brain, as well as high expression of Fam69C in the eye 60. Fam69A possesses a more ubiquitous distribution suggesting a specialized role for the Fam69B/C family members. In addition, Fam69B is upregulated in coordination with the pancreatic emergency response during pancreatitis 61.
Interestingly, members of the Fam69 family possess potential EF-hand calcium binding domains within the N-terminus of their kinase domain 59. This architectural rearrangement suggests that these proteins may be involved in calcium signaling within the brain, but no evidence has been presented yet.
Secreted Bacterial Kinases
Bacteria typically possess very few eukaryotic-like protein kinases. This is due, in part, to a heavy reliance on the two-component system, which utilizes sensory histidine kinases for communicating extracellular signals and environmental cues 62. However, bacteria do possess intracellular eukaryotic-like protein kinases (i.e PknB) 63. The bacterial secreted kinases are typically utilized as effector proteins that are injected into host organisms during bacterial pathogenicity. These kinases are often held inactive in the bacteria and are only activated within the host cytosol.
Pathogenic bacteria exist in a constant evolutionary arms race with their host organisms 64, 65. Thus, it is often advantageous for them to acquire new effector genes to modulate pathogenicity. These genes, often found on “pathogenicity islands”, are generally thought to have been acquired via horizontal gene transfer64. Sources for such genes can be found in other bacteria, parasites, and often in non-related eukaryotes. Closer inspection of the nearest homologs of many bacterial effector kinases reveal similarity with kinases found in eukarya. Phylogenetic analysis suggests that these effectors are eukaryotic-like because they can be contained within the standard human kinome tree with remote homology to canonical kinases such as CK1 and PKA (Figure 3). However, many other bacterial effector kinases show very little similarity to any known eukaryotic kinases, suggesting that they evolved divergently or their nearest neighbor has yet to be identified.
Bacteria can secrete proteins using any of 12 bacterial secretion systems found throughout the bacterial kingdom 66. A large portion of these systems are utilized in bacterial pathogenicity for injecting proteins into host organisms. These secretion complexes form channels that span the bacterial, and when necessary, host cell membranes. Proteins from the bacterial cytosol are localized to these systems based on signal sequences and binding of specific chaperones (Figure 4A). Effector proteins are injected either folded or unfolded 66 into the host cell where they usually carry out a specific function to aid in pathogenicity.
Of the known secreted bacterial pathogenic kinases, most of them can be grouped into two functional classes: 1) Those that participate in remodeling the host cytoskeleton and 2) Those that are involved in modulating immune signaling pathways in their hosts. Strikingly, so far, all identified kinases in bacteria are Ser/Thr kinases with no examples of Tyr kinases found yet.
Host Cytoskeletal Remodeling Ypka/SteC/LegK2
Several intracellular pathogens must remodel the host cell for successful disease progression. For example, Legionella and Salmonella both steal membranes from the host cell to create a vacuole (Legionella containing vacuole (LCV) and Salmonella containing vacuole (SCV)) in which they can grow and replicate. Yersinia infections are also commonly associated with cell rounding and cytoskeletal remodeling suggesting that Yersinia possess effectors involved in host remodeling. Thus, bacteria pirate host cell membrane and cytoskeleton signaling to achieve a replicative niche and aid in virulence. Several pathogen effector kinases participate in this process.
YpkA
The Yersinia protein kinase A (YpkA) was one of the first identified bacterial effector kinases. The Yersinia genus is comprised of three Gram-negative bacterial strains that are pathogenic to humans. The most infamous member of this genus is Yersinia pestis, (Y. pestis), the causative agent of the black plague. YpkA is an important virulence factor necessary for Yersinia pathogenicity and is secreted through a type-III secretion system (T3SS) into the host cell where it localizes to the inner plasma membrane 67. YpkA is activated upon actin binding and phosphorylates the Gαq heterotrimeric protein to inhibit guanine nucleotide binding (Figure 4B). This inhibition leads to disruption of Gαq signaling and ultimately disruption of actin filaments and cell rounding 68, 69. In an alternate but related pathway, YpkA also phosphorylates vasodilator stimulated phosphoprotein (VASP) and promotes actin skeletal rearrangements 70. This effect may be especially important in macrophages, which require actin remodeling to phagocytose bacterial cells.
SteC
SteC is a secreted eukaryotic-like kinase found in Salmonella. Upon uptake, Salmonella enterica serovar Typhimurium (S. Typhimurium) secretes various effectors to create a membrane bound vacuole, called the Salmonella containing vacuole (SCV). The integrity of the SCV is further reinforced by creation of an F-actin meshwork that surrounds the vesicles. SteC is secreted through a Salmonella Pathogenicity Island-2 Type-III Secretion System (SPI-2 T3SS) and is involved in F-actin meshwork formation. Bacterial strains lacking SteC successfully create the SCV but are defective in proper F-actin network organization 71. SteC specifically phosphorylates a residue in the inhibitory Helix A of the MAP kinase, MEK1, leading to structural rearrangements that allow its subsequent autophosphorylation and activation 72. MEK1 activation then signals through ERK and myosin light chain kinase (MLCK) ultimately culminating in Myosin IIB dependent actin cytoskeletal rearrangement in the vicinity of the SCV (Figure 4B). It has also been suggested that SteC is capable of inducing Myosin IIB independent actin remodeling through an unknown mechanism. Proper F-actin meshwork formation appears to restrict or enable replication of Salmonella within the SCV and thus regulate its virulence 72.
LegK2
Legionella pneumophila (L. pneumophila) replicates intracellularly in a Legionella containing vacuole (LCV) upon phagocytosis by macrophages. The LCV is a complex membranous organelle populated with numerous Legionella effectors that successfully allow initial escape from lysosomal degradation, followed by remodeling of the LCV membrane to subvert host immune responses and provide a replicative niche for the pathogen. Legionella possess a family of eukaryotic-like kinases, LegK1–4 that are potentially involved in this process.
LegK2 appears to be involved in host remodeling through inhibition of actin polymerization. Specifically, LegK2 localizes to the LCV and interacts with the ARPC1B and ARP3 subunits of the ARP2/3 complex. Phosphorylation of this complex results in inhibition of actin polymerization and endosomal/lysosomal fusion with the LCV 73 (Figure 4B). This may have a protective role for L. pneumophila by preventing degradation of their replicative vacuole.
Host Pathway Manipulation LegK1/OspG/NleH/LegK7
Evading and disrupting host cell immune signaling is an essential function for effector proteins. NF-κB is a central pathway involved in defense against microbes. Upon detection of pathogenic associated molecular patterns (PAMP’s) during infection, host cells activate NF-κB to control inflammation, cytokine production, and cell death 74. However, bacteria have evolved mechanisms to disrupt host NF-κB signaling 75. Three kinases have emerged that are capable of modulating host immune responses; LegK1, OspG, and NleH1. These proteins are found in strains of L. pneumophila, Shigella flexneri (S. flexneri), and enterohemorrhagic Escherichia coli (EHEC), respectively.
LegK1
L. pneumophila activates PAMP-dependent and T4SS dependent activation of NF-κB signaling 76. LegK1 was initially identified in an attempt to find T4SS factors capable of regulating NF-κB signaling. LegK1 activates NF-κB independent of the IKK complex through direct phosphorylation of the Iκβα subunit. Furthermore, LegK1 activates the non-canonical NF-κB pathway through phosphorylation of p100 (Figure 4C). Interestingly, LegK1 activity appears to be exclusive to NF-κB activation as MAPK and IFN pathways were not affected 77. Because macrophages are the primary cell targeted by L. pneumophila, it appears that LegK1 is involved in modulation of macrophage defense and inflammatory response.
Although LegK1 and LegK2 have been characterized, the substrates and function of LegK3 and LegK4 are unknown. However, a crystal structure of LegK4 has provided insight into the mechanism of action for these effector kinases. LegK4 possesses a core eukaryotic-like kinase domain that catalyzes phosphorylation through the canonical mechanism, although the residues involved in binding the nucleotide are atypical. LegK4 is present in an α-G helical coordinated dimer form currently not found in eukaryotic kinases. It also possesses a novel cap-domain at the amino-terminus as well as an alpha helical bundle in the C-terminus of the protein 78. Interestingly, this N-terminal cap bears structural similarity to the N-terminal extension of the nuclear factor κB-inducing kinase (NIK). The pre-formation of the ion-pair in the apo structure of LegK4 suggests that it exists in a constitutively active form that allows it to act independently of host-cell dependent activation. This is consistent with data from LegK1 suggesting that activation is not necessary for LegK1 dependent NF-κB activation.
OspG and NleH
OspG is one of ~20 effectors found in S. flexneri, the causative agent of shigellosis. Yeast two-hybrid screens identified numerous E2 enzymes as binding partners for OspG. Further analysis showed that OspG does not affect transfer of ubiquitin from E1 to E2 proteins, and that OspG specifically binds to ubiquitylated E2 proteins. One of the E2 enzymes identified was UbcH5b, an E2 involved in ubiquitination of IκBα. OspG was shown to inhibit the ubiquitin transfer of UbcH5b to pIκBα, thus attenuating NF-κB signaling in a kinase dependent manner 79 (Figure 4C). Moreover, cells infected with Shigella lacking OspG activity showed increased inflammatory response, implicating OspG as an important factor in reducing NF-κB dependent inflammation during infection. OspG specifically binds poly-ubiquitinated proteins and this binding stimulates its kinase activity 80.
NleH has high sequence similarity to OspG and also has a role in immune signaling. NleH1 appears to prevent IκBα ubiquitination and subsequent degradation (Figure 4C). It does not affect phosphorylation of the subunit, but may stabilize the phosphorylated form and prevent its conversion to the ubiquitinated form. Infection experiments with strains of EHEC deficient in NleH proteins showed lower levels of IκBα in host cells compared to wild type strains. NleH dependent inhibition of IκBα ubiquitination occurs in a kinase dependent manner. In mouse models, NleH deficient EHEC also showed higher serum levels of KC (IL-8 homolog) suggesting successful p50/p52 translocation to the nucleus and upregulation of response genes 81. While the role of NleH in suppressing NF-κB signaling by reducing IκBα degradation appears clear, no direct substrate of NleH has been identified. Studies suggest that it works upstream of p65, but no clear connection as to how IκBα phosphorylation stabilization is linked to NleH-dependent inhibition of ubiquitination has been made.
Another role for NleH has been suggested that is different than its close relative, OspG. Cells infected with EHEC lacking NleH are prone to undergo apoptosis 82. Using yeast two-hybrid screens, NleH was shown to bind the Bax inhibitor BI-1 and prevent activation and cleavage of procaspase 3 independent of its kinase activity 83. Thus, NleH may serve in both attenuated immune-signaling as well as inhibition of apoptosis in infected cells.
The crystal structures for both OspG and NleH have been solved (PDB: 4BVU, 4LRK, 4LRJ). Not surprisingly, both structures are highly similar. However, these kinases adopt a minimal fold lacking large portions of the C-lobe typically involved in substrate recognition and activation (Figure 3). Conservation of catalytic and metal-binding aspartates as well as the ion-pair suggest that these kinases undergo phospho-transfer utilizing the canonical mechanism.
LegK7
The newest member of the Legionella kinase repertoire is LegK7. LegK7 shows remote sequence similarity to any known eukaryotic kinase or the other four Legionella kinases. LegK7 mimics the MST1 kinase and phosphorylates host MOB1 during an infection to modulate Hippo signaling. Ultimately, LegK7 causes dysregulation of PPARγ genes through sequestration and degradation of the YAP1/TAZ complex in the cytoplasm. Intriguingly, Legionella replicate to a lesser extent when PPARγ is inhibited pharmacologically. However, knockout of LegK7 does not produce this phenotype, consistent with functional redundancy in the Legionella effector repertoire 84.
Secreted protozoan parasite kinases
Protozoan parasites include members of phylogenetically diverse supergroups including Excavata and Alveolata. As eukaryotes, they express typical eukaryotic Ser/Thr protein kinases, which include both conserved families such as AGC and CAMK, as well as lineage-specific kinase families that facilitate the organisms’ parasitic lifestyles. The most studied secreted parasite kinases are encoded by Apicomplexan parasites. The phylum Apicomplexa is comprised almost exclusively of obligate intracellular parasites that include the causative agents of some of the most widespread and devastating diseases worldwide, including malaria (Plasmodium spp.), cryptosporidiosis (Cryptosporidium spp.), and toxoplasmosis (Toxoplasma gondii).
Plasmodium falciparum kinases: The FIKK family
Plasmodium falciparum is the causative agent of the most severe forms of human malaria. Malaria disease symptoms are due to the asexual expansion of the parasite within the host’s erythrocytes. Infecting this cell type represents a unique challenge to the parasite; human red blood cells have a pared down complement of expressed proteins (several hundred) and no nucleus, so there are far fewer host proteins to manipulate in setting up a replicative niche. Plasmodium responds to this challenge by exporting its own effector proteins into the host cytosol, creating a complex trafficking and signaling system 85. Among these are an assortment of predicted secreted kinases, including members of the CMGC, Nek, and CK1 families as well as the parasite-specific FIKK family. PfCK1 is refractory to deletion, suggesting that it is essential, and has been reported to be exported to the erythrocyte cytoplasm, and may be additionally transported into the extracellular space (i.e. the human circulatory system) 86, though its function remains unknown.
The FIKK family was named based on the characteristic and unusual substitution of the residues Phe-Ile-Lys-Lys for the canonical VAIK motif, and is found only in Apicomplexan parasites. While most parasites have a single, intracellular FIKK kinase, the family is expanded in Plasmodium species to varied levels, with P. falciparum encoding 20 members of the family 87. These 19 expanded FIKK family members are predicted to be exported across the parasitophorous vacuole membrane into the host cell during infection 85. While no structure of a FIKK kinase has been solved, the kinases are highly divergent from human kinases 87, and therefore represent attractive drug targets. Indeed, loss of many of the FIKK kinases results in parasites with a severe loss of fitness in a genome-wide PiggyBAC screen 88. While the functions of the FIKK family members remain largely a mystery, two of the proteins, PfFIKK7.1 and PfFIKK12, have been implicated in modulating phosphorylation of the erythrocyte cytoskeleton upon infection 89. Knockouts for these two kinases are viable in culture, but infected cells have a modest reduction in membrane rigidity, which was used to suggest that these kinases may be important for protecting the parasitized blood cells from clearance from the host circulatory system. Similarly, loss of PfFIKK4.2 reduces both the rigidity and cytoadhesion of the infected erythrocyte 90, properties that are responsible for the most severe symptoms of P. falciparum malaria. Thus, the FIKK kinases may regulate both the parasite lytic cycle and the interaction of the host cell with its environment.
Toxoplasma gondii kinases: The ROPK family
Toxoplasma gondii is arguably the most successful parasite in the world, as it is able to infect virtually any warm-blooded animal. An estimated 30% of humans worldwide are infected with the parasite 91. Like Plasmodium, Toxoplasma survives inside an infected cell in a specialized organelle called the parasitophorous vacuole. Unlike Plasmodium, Toxoplasma exclusively infects nucleated cells, such as macrophages and neurons, and is estimated to use over 1000 effector proteins to co-opt host signaling and transcription. Remarkably, approximately one third of the parasite’s ~160 protein kinases are predicted to be secreted 92. These secreted effector kinases are largely comprised of another parasite specific family called the ROPK’s, which are named after the secretory organelle from which the majority of the proteins are secreted (the rhoptries). These kinases are remarkably diverse in sequence, with an average of <30% sequence identity between family members in the kinase domains. In addition, about half of the ROPK’s have substitutions in their active sites that are predicted to render them catalytically inactive 92, 93, making them the largest and most diverse family of pseudokinases yet described in Eukaryota. In addition, the genes encoding the ROPK’s (and the pseudokinases in particular), are often duplicated in tandem arrays, yielding fast-evolving loci that suggest a strong diversifying evolutionary pressure typical of host-pathogen interaction 93.
The two most ancient members of the ROPK family, ROP21 and ROP27, have recently been identified as secreted into the lumen of the parasitophorous vacuole, where they help mediate the transition of the parasite from the fast-growing acute stage to the chronic encysted stage of growth 94. All other described members of the ROPK family are secreted from the parasite’s rhoptries directly into the host cell’s cytoplasm during invasion 95. Not surprisingly, these kinases have been associated with striking alterations to the infected cell’s physiology that vary depending on the allele of the secreted kinase. For instance, allelic differences in ROP16 is associated with a ~10-fold variation in virulence in a mouse model of infection 96. Remarkably, a single substitution in ROP16 causes an allele that specifically and rapidly phosphorylates STAT3 97 and STAT6 98, bypassing typical JAK signaling in the activation of typical cytokine-induced immunomodulatory transcriptional profiles 96, 97. Remarkably, the ROP16 allele that activates STAT3/6 is protective to the host 96, and variation in this locus highlights one way in which different strains of the parasite may have evolved for success in diverse host niches.
The most potent strain-specific variation in virulence in mice is mediated by a partnership between two active ROPK’s, ROP17 and ROP18, as well as a family of pseudokinases collectively called ROP5. Together these proteins synergistically block the activation of the immune-related GTPases (IRGs) 99, 100 that are critical for the cell-autonomous control of intracellular pathogens. Remarkably, while individual knockouts for the active kinases ROP17 100 and ROP18 8, 100 show only a modest loss of virulence in the highly virulent RH strain, loss of the catalytically inactive ROP5 pseudokinase results in completely attenuated parasites 101. This is because ROP5 complexes with ROP17/ROP18 100 and simultaneously acts as a direct inhibitor of IRG-activating multimerization 99,102, thus preparing the IRGs for phosphorylation by its partner kinases.
The above examples comprise only 6 of the ~50 ROPKs, and it is clear we have only scratched the surface in our understanding of their diverse functions. For instance, another active ROPK, ROP38, has been associated with tempering global transcriptional changes due to infection by Toxoplasma 92 through an unknown mechanism, though it likely acts to regulate the functions of other parasite secreted effectors (which may not be kinases). In addition, given the recent unexpected catalytic functions of other pseudokinases 103, it is possible that a subset of the 20–30 predicted pseudokinases in the ROPK family may, themselves, possess non-phosphotransferase catalytic functions, which could reveal unusual regulatory mechanisms in the host human cells.
Concluding Remarks
Much is still to be discovered regarding the biological functions and consequences of the secreted protein kinases. Uncovering new atypical kinases in bacteria has opened the doorway to an expansion of the eukaryotic-like protein kinome into bacteria. The discovery of secreted kinases in humans has also allowed us to delve deeper into the mechanisms underlying various diseases and has challenged our canonical view of how a kinase functions. In addition, how these kinases work synergistically with phosphatases is poorly understood. Many secreted phosphatases, (secreted alkaline and acid phosphatases) that function on important secreted phosphoproteins are known. Diseases associated with these phosphatases share remarkable overlap with diseases associated with kinases such as skeletal and neuronal defects 104–106. There is much to explore and the possibilities of discovering new and even more radical kinases is an exciting prospect with potential to unearth entirely new fields of study.
Acknowledgements
We thank members of the Tagliabracci laboratory for insightful discussions. Work in the authors laboratory is supported by NIH Grants R00DK099254 (V.S.T.), Welch Foundation Grants I-1911 (V.S.T), I-1936 (M.L.R), a CPRIT grant RP170674 (V.S.T), and an NSF grant MCB1553334 (M.L.R). V.S.T. is the Michael L. Rosenberg Scholar in Medical Research, Cancer Prevention Research Institute of Texas Scholar (RR150033) and Searle Scholar.
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