Male Germ Cell-associated Kinase Research Articles

First person – Hung-Ju Chiang

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Disease Models & Mechanisms, helping researchers promote themselves alongside their papers. Hung-Ju Chiang is first author on ‘ Male germ cell-associated kinase is required for axoneme formation during ciliogenesis in zebrafish photoreceptors’, published in DMM. Hung-Ju is a Junior Research Fellow in the lab of Ichiro Masai at Okinawa Institute of Science and Technology Graduate University, Japan, investigating the importance of cilia, a largely underappreciated organelle.

Male germ cell-associated kinase is required for axoneme formation during ciliogenesis in zebrafish photoreceptors.

Vertebrate photoreceptors are highly specialized retinal neurons that have cilium-derived membrane organelles called outer segments, which function as platforms for phototransduction. Male germ cell-associated kinase (MAK) is a cilium-associated serine/threonine kinase, and its genetic mutation causes photoreceptor degeneration in mice and retinitis pigmentosa in humans. However, the role of MAK in photoreceptors is not fully understood. Here, we report that zebrafish mak mutants show rapid photoreceptor degeneration during embryonic development. In mak mutants, both cone and rod photoreceptors completely lacked outer segments and underwent apoptosis. Interestingly, zebrafish mak mutants failed to generate axonemes during photoreceptor ciliogenesis, whereas basal bodies were specified. These data suggest that Mak contributes to axoneme development in zebrafish, in contrast to mouse Mak mutants, which have elongated photoreceptor axonemes. Furthermore, the kinase activity of Mak was found to be critical in ciliary axoneme development and photoreceptor survival. Thus, Mak is required for ciliogenesis and outer segment formation in zebrafish photoreceptors to ensure intracellular protein transport and photoreceptor survival.

Development of gene augmentation strategy for the treatment of MAK associated Retinitis Pigmentosa

PurposeThe gene male germ cell associated kinase (MAK) encodes a protein involved in regulation of photoreceptor cilia length. By combining next generation whole exome sequencing and induced pluripotent stem cell (iPSC) technology we found that an Alu repeat inserted in exon 9 of the MAK gene resulted in a loss of normal MAK transcript and development of human autosomal recessive retinitis pigmentosa (RP). Although a relatively rare cause of disease in the general population, the MAK variant is enriched in individuals of Ashkenazi Jewish descent. In this population, 1 in 55 individuals are carriers and 1/3 of all cases of recessive RP is caused by this gene. The purpose of this study was to determine if a viral gene augmentation strategy could be used to restore functional MAK protein and in turn would be a good candidate for treatment of early stage MAK associated RP.MethodsTo demonstrate the ability of the developed MAK gene transfer vector to restore normal transcript and protein, patient specific iPSC derived photoreceptor precursor cells were transduced and immunocytochemistry, rt‐PCR and western blot analysis were performed. To demonstrate transgene function, cilia length assays were performed using patient derived fibroblast cells in vitro and MAK knock out zebra fish in vivo. In addition, visual function testing was performed in the zebra fish. To demonstrate safety of cGMP generated clinical grade vector, local and systemic toxicity studies were performed via subretinal injection into wildtype rats.ResultsMAK mutant RP iPSC derived photoreceptor cells harboring the previously identified ALU insertion (Tucker et. al. 2011) were generated and transduced with viral vectors containing the retinal MAK transcript. At 1‐week post‐transduction normal retinal MAK transcript and protein could be detected via rt‐PCR and western blotting respectively. Using patient derived fibroblast cells and MAK knockdown zebra fish we demonstrate that over expression of retinal MAK transgene restored primary cilia length. In addition, the visual defect identified in MAK knockdown zebra fish was mitigated via treatment with retinal MAK transgene. Following subretinal delivery of clinical grade vector into wild type rats no evidence of local or systemic toxicity was detected at either 1 or 3 months' post‐treatment.ConclusionsWe have successfully developed a MAK gene replacement strategy that has been validated in human iPSC derived photoreceptor precursor cells in vitro and MAK knock down zebra fish and wildtype rats in vivo.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The kinases male germ cell-associated kinase and cell cycle-related kinase regulate kinesin-2 motility in Caenorhabditis elegans neuronal cilia.

Kinesin-2 motors power anterograde intraflagellar transport (IFT), a highly ordered process that assembles and maintains cilia. However, it remains elusive how kinesin-2 motors are regulated in vivo. Here, we performed forward genetic screens to isolate suppressors that rescue the ciliary defects of OSM-3-kinesin (homolog of mammalian homodimeric kinesin-2 KIF17) mutants in Caenorhabditis elegans. We identified the C. elegans dyf-5 and dyf-18, which encode the homologs of mammalian male germ cell-associated kinase and cell cycle-related kinase, respectively. Using time-lapse fluorescence microscopy, we show that DYF-5 and DYF-18 are IFT cargo molecules and are enriched at the distal segments of sensory cilia. Mutations of dyf-5 and dyf-18 generate elongated cilia and ectopic localization of the heterotrimeric kinesin-2 (kinesin-II) at the ciliary distal segments. Genetic analyses reveal that dyf-5 and dyf-18 are important for stabilizing the interaction between IFT particles and OSM-3-kinesin. Our data suggest that DYF-5 and DYF-18 act in the same pathway to promote handover between kinesin-II and OSM-3 in sensory cilia.

Nonsyndromic Retinitis Pigmentosa in the Ashkenazi Jewish Population: Genetic and Clinical Aspects

To analyze the genetic and clinical findings in retinitis pigmentosa (RP) patients of Ashkenazi Jewish (AJ) descent, aiming to identify genotype-phenotype correlations. Cohort study. Retinitis pigmentosa patients from 230 families of AJ origin. Sanger sequencing was performed to detect specific founder mutations known to be prevalent in the AJ population. Ophthalmologic analysis included a comprehensive clinical examination, visual acuity (VA), visual fields, electroretinography, color vision testing, and retinal imaging by OCT, pseudocolor, and autofluorescence fundus photography. Inheritance pattern and causative mutation; retinal function as assessed by VA, visual fields, and electroretinography results; and retinal structural changes observed on clinical funduscopy as well as by pseudocolor, autofluorescence, and OCT imaging. The causative mutation was identified in 37% of families. The most prevalent RP-causing mutations are the Alu insertion (c.1297_8ins353, p.K433Rins31*) in the male germ cell-associated kinase (MAK) gene (39% of families with a known genetic cause for RP) and c.124A>G, p.K42E in dehydrodolichol diphosphate synthase (DHDDS) (33%). Additionally, disease-causing mutations were identified in 11 other genes. Analysis of clinical parameters of patients with mutations in the 2 most common RP-causing genes revealed that MAK patients had better VA and visual fields at relatively older ages in comparison with DHDDS patients. Funduscopic findings of DHDDS patients matched those of MAK patients who were 20 to 30 years older. Patients with DHDDS mutations were referred for electrophysiologic evaluation at earlier ages, and their cone responses became nondetectable at a much younger age than MAK patients. Our AJ cohort of RP patients is the largest reported to date and showed a substantial difference in the genetic causes of RP compared with cohorts of other populations, mainly a high rate of autosomal recessive inheritance and a unique composition of causative genes. The most common RP-causing genes in our cohort, MAK and DHDDS, were not described as major causative genes in other populations. The clinical data show that in general, patients with biallelic MAK mutations had a later age of onset and a milder retinal phenotype compared with patients with biallelic DHDDS mutations.

Using CRISPR-Cas9 to Generate Gene-Corrected Autologous iPSCs for the Treatment of Inherited Retinal Degeneration

Patient-derived induced pluripotent stem cells (iPSCs) hold great promise for autologous cell replacement. However, for many inherited diseases, treatment will likely require genetic repair pre-transplantation. Genome editing technologies are useful for this application. The purpose of this study was to develop CRISPR-Cas9-mediated genome editing strategies to target and correct the three most common types of disease-causing variants in patient-derived iPSCs: (1) exonic, (2) deep intronic, and (3) dominant gain of function. We developed a homology-directed repair strategy targeting a homozygous Alu insertion in exon 9 of male germ cell-associated kinase (MAK) and demonstrated restoration of the retinal transcript and protein in patient cells. We generated a CRISPR-Cas9-mediated non-homologous end joining (NHEJ) approach to excise a major contributor to Leber congenital amaurosis, the IVS26 cryptic-splice mutation in CEP290, and demonstrated correction of the transcript and protein in patient iPSCs. Lastly, we designed allele-specific CRISPR guides that selectively target the mutant Pro23His rhodopsin (RHO) allele, which, following delivery to both patient iPSCs invitro and pig retina invivo, created a frameshift and premature stop that would prevent transcription of the disease-causing variant. The strategies developed in this study will prove useful for correcting a wide range of genetic variants in genes that cause inherited retinal degeneration.

Efficient In Silico Identification of a Common Insertion in the MAK Gene which Causes Retinitis Pigmentosa.

BackgroundNext generation sequencing (NGS) offers a rapid and comprehensive method of screening for mutations associated with retinitis pigmentosa and related disorders. However, certain sequence alterations such as large insertions or deletions may remain undetected using standard NGS pipelines. One such mutation is a recently-identified Alu insertion into the Male Germ Cell-Associated Kinase (MAK) gene, which is missed by standard NGS-based variant callers. Here, we developed an in silico method of searching NGS raw sequence reads to detect this mutation, without the need to recalculate sequence alignments or to screen every sample by PCR.MethodsThe Linux program grep was used to search for a 23 bp “probe” sequence containing the known junction sequence of the insert. A corresponding search was performed with the wildtype sequence. The matching reads were counted and further compared to the known sequences of the full wildtype and mutant genomic loci. (See https://github.com/MEEIBioinformaticsCenter/grepsearch.)ResultsIn a test sample set consisting of eleven previously published homozygous mutants, detection of the MAK-Alu insertion was validated with 100% sensitivity and specificity. As a discovery cohort, raw NGS reads from 1,847 samples (including custom and whole exome selective capture) were searched in ~1 hour on a local computer cluster, yielding an additional five samples with MAK-Alu insertions and solving two previously unsolved pedigrees. Of these, one patient was homozygous for the insertion, one compound heterozygous with a missense change on the other allele (c. 46G>A; p.Gly16Arg), and three were heterozygous carriers.ConclusionsUsing the MAK-Alu grep program proved to be a rapid and effective method of finding a known, disease-causing Alu insertion in a large cohort of patients with NGS data. This simple approach avoids wet-lab assays or computationally expensive algorithms, and could also be used for other known disease-causing insertions and deletions.

Transcriptomic analysis across nasal, temporal, and macular regions of human neural retina and RPE/choroid by RNA-Seq

Proper spatial differentiation of retinal cell types is necessary for normal human vision. Many retinal diseases, such as Best disease and male germ cell associated kinase (MAK)-associated retinitis pigmentosa, preferentially affect distinct topographic regions of the retina. While much is known about the distribution of cell types in the retina, the distribution of molecular components across the posterior pole of the eye has not been well-studied. To investigate regional difference in molecular composition of ocular tissues, we assessed differential gene expression across the temporal, macular, and nasal retina and retinal pigment epithelium (RPE)/choroid of human eyes using RNA-Seq. RNA from temporal, macular, and nasal retina and RPE/choroid from four human donor eyes was extracted, poly-A selected, fragmented, and sequenced as 100 bp read pairs. Digital read files were mapped to the human genome and analyzed for differential expression using the Tuxedo software suite. Retina and RPE/choroid samples were clearly distinguishable at the transcriptome level. Numerous transcription factors were differentially expressed between regions of the retina and RPE/choroid. Photoreceptor-specific genes were enriched in the peripheral samples, while ganglion cell and amacrine cell genes were enriched in the macula. Within the RPE/choroid, RPE-specific genes were upregulated at the periphery while endothelium associated genes were upregulated in the macula. Consistent with previous studies, BEST1 expression was lower in macular than extramacular regions. The MAK gene was expressed at lower levels in macula than in extramacular regions, but did not exhibit a significant difference between nasal and temporal retina. The regional molecular distinction is greatest between macula and periphery and decreases between different peripheral regions within a tissue. Datasets such as these can be used to prioritize candidate genes for possible involvement in retinal diseases with regional phenotypes.

In silico 3D structure modeling and inhibitor binding studies of human male germ cell-associated kinase

Human male germ cell-associated kinase (hMAK) is an androgen-inducible gene in prostate epithelial cells, and it acts as a coactivator of androgen receptor signaling in prostate cancer. The 3D structure of the hMAK kinase was modeled based on the crystal structure of CDK2 kinase using comparative modeling methods, and the ATP-binding site was characterized. We have collected five inhibitors of hMAK from the literature and docked into the ATP-binding site of the kinase domain. Solvated interaction energies (SIE) of inhibitor binding are calculated from the molecular dynamics simulations trajectories of protein–inhibitor complexes. The contribution from each active site residue in hMAK toward inhibitor binding revealed the nature and extent of interactions between inhibitors and individual residues. The main chain atoms of Met79 invariably form hydrogen bonds with all five inhibitors. The amino acids Leu7, Val15, and Leu129 stabilize the inhibitors via CH–pi interactions. The Asp140 in the active site and Glu77 in hinge region show characteristic hydrogen bonding interactions with inhibitors. From SIE, the residue-wise interactions revealed the nature of non-bonding contacts and modifications required to increase the inhibitor activity. Our work provides 3D model structure of hMAK and molecular basis for the mechanisms of hMAK inhibition at atomic level that aid in designing new potent inhibitors.

Distinct Expression Patterns of ICK/MAK/MOK Protein Kinases in the Intestine Implicate Functional Diversity

ICK/MRK (intestinal cell kinase/MAK-related kinase), MAK (male germ cell-associated kinase), and MOK (MAPK/MAK/MRK-overlapping kinase) are closely related serine/threonine protein kinases in the protein kinome. The biological functions and regulatory mechanisms of the ICK/MAK/MOK family are still largely elusive. Despite significant similarities in their catalytic domains, they diverge markedly in the sequence and structural organization of their C-terminal non-catalytic domains, raising the question as to whether they have distinct, overlapping, or redundant biological functions. In order to gain insights into their biological activities and lay a fundamental groundwork for functional studies, we investigated the spatio-temporal distribution patterns and the expression dynamics of ICK/MAK/MOK protein kinases in the intestine. We found that ICK/MAK/MOK proteins display divergent expression patterns along the duodenum-to-colon axis and during postnatal murine development. Furthermore, they are differentially partitioned between intestinal epithelium and mesenchyme. A significant increase in the protein level of ICK, but not MAK, was induced in human primary colon cancer specimens. ICK protein level was up-regulated whereas MOK protein level was down-regulated in mouse intestinal adenomas as compared with their adjacent normal intestinal mucosa. These data suggest distinct roles for ICK/MAK/MOK protein kinases in the regulation of intestinal neoplasia. Taken together, our findings demonstrate that the expressions of ICK/MAK/MOK proteins in the intestinal tract can be differentially and dynamically regulated, implicating a significant functional diversity within this group of protein kinases.

Regulation of cilium length and intraflagellar transport by the MAP kinases MAK, MRK, and MOK

Cilia are assembled and maintained by intraflagellar transport (IFT), which moves cargo bi-directionally along the axoneme of the cilium. Regulation of IFT allows modulation of cilia length and influences signaling pathways that initiate in the cilium. We characterized C. elegans’ DYF-5, a MAP kinase that regulates cilia length and IFT. DYF-5 has three mammalian homologues: MAK (male germ cell-associated kinase), MRK (MAK-related kinase) and MOK (MAPK/MAK/MRK overlapping kinase). Here we analyzed the functions of MAK, MRK and MOK. GFP fusions of MAK, MRK and MOK showed that all three proteins localize along the length of the cilia of IMCD3 and RPE cells, and accumulate at the distal tip. Knockdown of MRK or MOK resulted in longer cilia, while overexpression of MAK or MRK resulted in short and stumpy cilia. Interestingly, overexpression of MOK did not affect cilia length. These effects on cilia length are in line with our data on DYF-5 function in C. elegans (Burghoorn et al., 2007) and published data on MAK (Omori et al., 2010). To visualize IFT and measure its speed we have generated IMCD3 cell lines that stably express GFP-fusion constructs of different components of the IFT complex: Kinesin-II, Kif17, complex A, complex B, and the BBSome. All these GFP fusion proteins showed an average anterograde speed of ~0.4 μm/s and a similar average retrograde speed. Knock down of MOK using shRNAs resulted in increased anterograde speeds of all constructs to ~0.6 μm/s, while knock down of MRK had no effect. Retrograde speeds were not affected.

Different protein expression patterns associated with polycystic ovary syndrome in human follicular fluid during controlled ovarian hyperstimulation

Polycystic ovary syndrome (PCOS) is one of the most common causes of anovulatory infertility, affecting 5-10% of females during their reproductive life. Currently the pathology of PCOS is largely unknown. To identify the differential protein expression in follicular fluids from PCOS and normal subjects during controlled ovarian hyperstimulation, we performed an initial proteomic study including two-dimensional gel electrophoresis (2DE) analysis and mass spectroscopy, and confirmed results by western blot. Thirty-two protein spots were shown to be significantly differentially expressed between PCOS and normal follicular fluids, of which 20 unique proteins were identified to be associated with cellular metabolism and physiological processes; 13 of these proteins were upregulated while seven were downregulated in PCOS follicular fluids. Western blotting analyses confirmed the differential expressions for three randomly selected proteins, i.e. upregulated α1-antitrypsin, apolipoprotein A-I and transferrin in follicular fluid from PCOS patients than normal controls. Furthermore, semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses revealed that mRNA levels of serine palmitoyltransferase 2, serine/threonine-protein kinase male germ cell-associated kinase (MAK) and DNA damage-regulated autophagy modulator protein 2 decreased significantly in granulosa cells of PCOS patients compared with normal samples. These results increase our understanding of PCOS and the identified genes may serve as candidate biomarkers to develop diagnostic and therapeutic tools.

Autosomal Recessive Retinitis Pigmentosa Caused by Mutations in theMAKGene

To determine the disease expression in autosomal recessive (ar) retinitis pigmentosa (RP) caused by mutations in the MAK (male germ cell-associated kinase) gene. Patients with RP and MAK gene mutations (n = 24; age, 32-77 years at first visit) were studied by ocular examination, perimetry, and optical coherence tomography (OCT). All but one MAK patient were homozygous for an identical truncating mutation in exon 9 and had Ashkenazi Jewish heritage. The carrier frequency of this mutation among 1207 unrelated Ashkenazi control subjects was 1 in 55, making it the most common cause of heritable retinal disease in this population and MAK-associated RP the sixth most common Mendelian disease overall in this group. Visual acuities could be normal into the eighth decade of life. Kinetic fields showed early loss in the superior-temporal quadrant. With more advanced disease, superior and midperipheral function was lost, but the nasal field remained. Only a central island was present at late stages. Pigmentary retinopathy was less prominent in the superior nasal quadrant. Rod-mediated vision was abnormal but detectable in the residual field; all patients had rod>cone dysfunction. Photoreceptor layer thickness was normal centrally but decreased with eccentricity. At the stages studied, there was no evidence of photoreceptor ciliary elongation. The patterns of disease expression in the MAK form of arRP showed some resemblance to patterns described in autosomal dominant RP, especially the form caused by RP1 mutations. The similarity in phenotypes is of interest, considering that there is experimental evidence of interaction between Mak and RP1 in the photoreceptor cilium.

Male germ cell-associated kinase is overexpressed in prostate cancer cells and causes mitotic defects via deregulation of APC/CCDH1

Male germ cell-associated kinase (MAK), a direct transcriptional target of androgen receptor (AR), is a co-activator of AR. In this study, we determined the activating mechanism of MAK and identified a previously unknown AR-independent role of MAK in mitosis. We found that MAK kinase activity requires dual phosphorylation of the conserved TDY motif and that the phosphorylation is dynamic during cell cycle. MAK associates with CDH1 (FZR1, fizzy/cell division cycle 20 related 1) and phosphorylates CDH1 at sites phosphorylated by cyclin-dependent kinases. When MAK is overexpressed, the binding of CDH1 to anaphase promoting complex/cyclosome decreased, resulting in an attenuation of anaphase-promoting complex/C ubiquitin ligase activity and the consequential stabilization of the CDH1 targets such as Aurora kinase A and Polo-like kinase 1. As such, overexpression of MAK leads to mitotic defects such as centrosome amplification and lagging chromosomes. Our immunohistochemistry result showed that MAK is overexpressed in prostate tumor tissues, suggesting a role of MAK in prostate carcinogenesis. Taken with our previous results, our data implicate MAK in both AR activation and chromosomal instability, acting in both early and late prostate cancer development.

Exome sequencing and analysis of induced pluripotent stem cells identify the cilia-related gene male germ cell-associated kinase ( MAK ) as a cause of retinitis pigmentosa

Retinitis pigmentosa (RP) is a genetically heterogeneous heritable disease characterized by apoptotic death of photoreceptor cells. We used exome sequencing to identify a homozygous Alu insertion in exon 9 of male germ cell-associated kinase (MAK) as the cause of disease in an isolated individual with RP. Screening of 1,798 unrelated RP patients identified 20 additional probands homozygous for this insertion (1.2%). All 21 affected probands are of Jewish ancestry. MAK encodes a kinase involved in the regulation of photoreceptor-connecting cilium length. Immunohistochemistry of human donor tissue revealed that MAK is expressed in the inner segments, cell bodies, and axons of rod and cone photoreceptors. Several isoforms of MAK that result from alternative splicing were identified. Induced pluripotent stem cells were derived from the skin of the proband and a patient with non-MAK-associated RP (RP control). In the RP control individual, we found that a transcript lacking exon 9 was predominant in undifferentiated cells, whereas a transcript bearing exon 9 and a previously unrecognized exon 12 predominated in cells that were differentiated into retinal precursors. However, in the proband with the Alu insertion, the developmental switch to the MAK transcript bearing exons 9 and 12 did not occur. In addition to showing the use of induced pluripotent stem cells to efficiently evaluate the pathogenicity of specific mutations in relatively inaccessible tissues like retina, this study reveals algorithmic and molecular obstacles to the discovery of pathogenic insertions and suggests specific changes in strategy that can be implemented to more fully harness the power of sequencing technologies.

Exome Sequencing and cis-Regulatory Mapping Identify Mutations in MAK, a Gene Encoding a Regulator of Ciliary Length, as a Cause of Retinitis Pigmentosa

A fundamental challenge in analyzing exome-sequence data is distinguishing pathogenic mutations from background polymorphisms. To address this problem in the context of a genetically heterogeneous disease, retinitis pigmentosa (RP), we devised a candidate-gene prioritization strategy called cis-regulatory mapping that utilizes ChIP-seq data for the photoreceptor transcription factor CRX to rank candidate genes. Exome sequencing combined with this approach identified a homozygous nonsense mutation in male germ cell-associated kinase (MAK) in the single affected member of a consanguineous Turkish family with RP. MAK encodes a cilium-associated mitogen-activated protein kinase whose function is conserved from the ciliated alga, Chlamydomonas reinhardtii, to humans. Mutations in MAK orthologs in mice and other model organisms result in abnormally long cilia and, in mice, rapid photoreceptor degeneration. Subsequent sequence analyses of additional individuals with RP identified five probands with missense mutations in MAK. Two of these mutations alter amino acids that are conserved in all known kinases, and an invitro kinase assay indicates that these mutations result in a loss of kinase activity. Thus, kinase activity appears to be critical for MAK function in humans. This study highlights a previously underappreciated role for CRX as a direct transcriptional regulator of ciliary genes in photoreceptors. In addition, it demonstrates the effectiveness of CRX-based cis-regulatory mapping in prioritizing candidate genes from exome data and suggests that this strategy should be generally applicable to a range of retinal diseases.

Negative regulation of ciliary length by ciliary male germ cell-associated kinase (Mak) is required for retinal photoreceptor survival

Cilia function as cell sensors in many organs, and their disorders are referred to as "ciliopathies." Although ciliary components and transport machinery have been well studied, regulatory mechanisms of ciliary formation and maintenance are poorly understood. Here we show that male germ cell-associated kinase (Mak) regulates retinal photoreceptor ciliary length and subcompartmentalization. Mak was localized both in the connecting cilia and outer-segment axonemes of photoreceptor cells. In the Mak-null retina, photoreceptors exhibit elongated cilia and progressive degeneration. We observed accumulation of intraflagellar transport 88 (IFT88) and IFT57, expansion of kinesin family member 3A (Kif3a), and acetylated α-tubulin signals in the Mak-null photoreceptor cilia. We found abnormal rhodopsin accumulation in the Mak-null photoreceptor cell bodies at postnatal day 14. In addition, overexpression of retinitis pigmentosa 1 (RP1), a microtubule-associated protein localized in outer-segment axonemes, induced ciliary elongation, and Mak coexpression rescued excessive ciliary elongation by RP1. The RP1 N-terminal portion induces ciliary elongation and increased intensity of acetylated α-tubulin labeling in the cells and is phosphorylated by Mak. These results suggest that Mak is essential for the regulation of ciliary length and is required for the long-term survival of photoreceptors.

Proteomics Evaluation of Chemically Cleavable Activity-based Probes

Activity-based probes (ABPs) that specifically target subsets of related enzymatic proteins are finding increasing use in proteomics research. One of the main applications for these reagents is affinity isolation of probe-labeled targets. However, the use of cheap and efficient biotin affinity tags on ABPs can be problematic due to difficulty in release of captured proteins. Here we describe the evaluation of activity-based probes carrying a chemically cleavable linker that allows selective release of probe-labeled proteins under mild elution conditions that are compatible with mass spectrometric analysis. Specifically, we compare results from standard on-bead digestion of probe-labeled targets after affinity purification with the results obtained using chemoselective cleavage. Results are presented for multiple APBs that target both serine and cysteine proteases. These results highlight significant improvements in the quality of data obtained by using the cleavable linker system.

Identification of Yin-Yang Regulators and a Phosphorylation Consensus for Male Germ Cell-Associated Kinase (MAK)-Related Kinase

MAK (male germ cell-associated protein kinase) and MRK/ICK (MAK-related kinase/intestinal cell kinase) are human homologs of Ime2p in Saccharomyces cerevisiae and of Mde3 and Pit1 in Schizosaccharomyces pombe and are similar to human cyclin-dependent kinase 2 (CDK2) and extracellular signal-regulated kinase 2 (ERK2). MAK and MRK require dual phosphorylation in a TDY motif catalyzed by an unidentified human threonine kinase and tyrosine autophosphorylation. Herein, we establish that human CDK-related kinase CCRK (cell cycle-related kinase) is an activating T157 kinase for MRK, whereas active CDK7/cyclin H/MAT1 complexes phosphorylate CDK2 but not MRK. Protein phosphatase 5 (PP5) interacts with MRK in a complex and dephosphorylates MRK at T157 in vitro and in situ. Thus, CCRK and PP5 are yin-yang regulators of T157 phosphorylation. To determine a substrate consensus, we screened a combinatorial peptide library with active MRK. MRK preferentially phosphorylates R-P-X-S/T-P sites, with the preference for arginine at position -3 (P-3) being more stringent than for prolines at P-2 and P+1. Using the consensus, we identified a putative phosphorylation site (RPLT(1080)S) for MRK in human Scythe, an antiapoptotic protein that interacts with MRK. MRK phosphorylates Scythe at T1080 in vitro as determined by site-directed mutagenesis and mass spectrometry, supporting the consensus and suggesting Scythe as a physiological substrate for MRK.

Male Germ Cell–Associated Kinase, a Male-Specific Kinase Regulated by Androgen, Is a Coactivator of Androgen Receptor in Prostate Cancer Cells

Androgen receptor (AR) is a ligand-induced transcriptional factor, which plays an important role in the normal development of prostate as well as in the progression of prostate cancer. Numerous coactivators, which associate with AR and function to remodel chromatin and recruit RNA polymerase II to enhance the transcriptional potential of AR, have been identified. Among these coactivators, few are protein kinases. In this study, we describe the characterization of a novel protein kinase, male germ cell-associated kinase (MAK), which serves as a coactivator of AR. We present evidence, which indicates that (a) MAK physically associates with AR (MAK and AR are found to be coprecipitated from cell extracts, colocalized in nucleus, and corecruited to prostate-specific antigen promoter in LNCaP as well as in transfected cells); (b) MAK is able to enhance AR transactivation potential in an androgen- and kinase-dependent manner in several prostate cancer cells and synergize with ACTR/steroid receptor coactivator-3 coactivator; (c) small hairpin RNA (shRNA) knocks down MAK expression resulting in the reduction of AR transactivation ability; (d) MAK-shRNA or kinase-dead mutant, when introduced into LNCaP cells, reduces the growth of the cells; and (e) microarray analysis of LNCaP cells carrying kinase-dead MAK mutant showed a significant impediment of AR signaling, indicating that endogenous MAK plays a general role in AR function in prostate cancer cells and likely to be a general coactivator of AR in prostate tissues. The highly restricted expression of this kinase makes it a potentially useful target for intervention of androgen independence.