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.

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Intestinal cell kinase (ICK), originally cloned from the intestine and expressed in the intestinal crypt epithelium, is a highly conserved serine/threonine protein kinase that is similar to mitogen-activated protein kinases (MAPKs) in the catalytic domain and requires dual phosphorylation within a MAPK-like TDY motif for full activation. Despite these similarities to MAPKs, the biological functions of ICK remain unknown. In this study, we report that suppression of ICK expression in cultured intestinal epithelial cells by short hairpin RNA (shRNA) interference significantly impaired cellular proliferation and induced features of gene expression characteristic of colonic or enterocytic differentiation. Downregulation of ICK altered expression of cell cycle regulators (cyclin D1, c-Myc, and p21(Cip1/WAF1)) of G(1)-S transition, consistent with the G(1) cell cycle delay induced by ICK shRNA. ICK deficiency also led to a significant decrease in the expression and/or activity of p70 ribosomal protein S6 kinase (S6K1) and eukaryotic initiation factor 4E (eIF4E), concomitant with reduced expression of their upstream regulators, the mammalian target of rapamycin (mTOR) and the regulatory associated protein of mTOR (Raptor). Furthermore, ICK interacts with the mTOR/Raptor complex in vivo and phosphorylates Raptor in vitro. These results suggest that disrupting ICK function may downregulate protein translation of specific downstream targets of eIF4E and S6K1 such as cyclin D1 and c-Myc through the mTOR/Raptor signaling pathway. Taken together, our findings demonstrate an important role for ICK in proliferation and differentiation of intestinal epithelial cells.

Malnutrition and cryptosporidiosis form a vicious cycle and lead to acute and long-term growth impairment in children from developing countries. Insights into mechanisms underlying the vicious cycle will help to design rational therapies to mitigate this infection. We tested the effect of short-term protein malnutrition on Cryptosporidium parvum infection in a murine model by examining stool shedding, tissue burden, and histologic change and explored the mechanism underlying the interaction between malnutrition and cryptosporidiosis through immunostaining and immunoblotting. Protein malnutrition increased stool shedding and the number of intestine-associated C. parvum organisms, accompanied by significant suppression of C. parvum-induced caspase 3 activity and expression of PCNA and Ki67, but activation of the Akt survival pathway in intestinal epithelial cells. We find that even very brief periods of protein malnutrition may enhance (or intensify) cryptosporidiosis by suppressing C. parvum-induced cell turnover and caspase-dependent apoptosis of intestinal epithelial cells. This implicates a potential strategy to attenuate C. parvum's effects by modulating apoptosis and promoting regeneration in the intestinal epithelium.

Human endocrine-cerebro-osteodysplasia (ECO) syndrome, caused by the loss-of-function mutation R272Q in the ICK (intestinal cell kinase) gene, is a neonatal-lethal developmental disorder. To elucidate the molecular basis of ECO syndrome, we constructed an Ick R272Q knock-in mouse model that recapitulates ECO pathological phenotypes. Newborns bearing Ick R272Q homozygous mutations die at birth due to respiratory distress. Ick mutant lungs exhibit not only impaired branching morphogenesis associated with reduced mesenchymal proliferation, but also significant airspace deficiency in primitive alveoli concomitant with abnormal interstitial mesenchymal differentiation. ICK dysfunction induces elongated primary cilia and perturbs ciliary Hedgehog signaling and autophagy during lung sacculation. Our study identifies an essential role for ICK in lung development and advances the mechanistic understanding of ECO syndrome.

Loss-of-function mutations in the human ICK (intestinal cell kinase) gene cause dysfunctional primary cilia and perinatal lethality which are associated with human ciliopathies. The enzyme that we herein call CAPK (ciliopathy-associated protein kinase) is a serine/threonine protein kinase that has a highly conserved MAPK-like N-terminal catalytic domain and an unstructured C-terminal domain (CTD) whose functions are completely unknown. In this study, we demonstrate that truncation of the CTD impairs the ability of CAPK to interact with and phosphorylate its substrate, kinesin family member 3A (KIF3A). We also find that deletion of the CTD of CAPK compromises both localization to the primary cilium and negative regulation of ciliogenesis. Thus, CAPK substrate recognition, ciliary targeting, and ciliary function depend on the non-catalytic CTD of the protein which is predicted to be intrinsically disordered.

Objective: To investigate the association between semaphorin 3A (SEMA 3A) and its receptor neuropilin 1 (NRP1) and the clinicopathologic characteristics of patients with tongue cancer. Study Design: Forty-three tongue squamous cell carcinoma specimens were included. Immunohistochemical staining of SEMA3A and NRP1 was performed on 15 normal tongue epithelium specimens and the 43 tumour specimens. Immunoreactivity was evaluated based on the staining intensity and distribution score. Statistical analyses were performed using Chi-squared and Spearman tests and Kaplan-Meier analysis. Results: SEMA3A was significantly down-regulated in tongue cancer compared with normal tongue (P=0.025), while NRP1 was over-expressed in tumours (P<0.001). SEMA3A expression inversely correlated with nodal metastasis (P=0.017). NRP1 expression did not correlate with any clinicopathological characteristics. Higher SEMA3A expression strongly predicted longer survival (P=0.005). Scores for the NRP1/SEMA3A ratio of ≥1 predicted shorter survival (P=0.045). Conclusions: Aberrant expression of SEMA3A and its receptor NRP1 might be involved in the development of tongue cancer and might be useful prognostic markers in this tumour type. Key words:Semaphorin 3A, neuropilin 1, tongue, squamous cell carcinoma.

Background:Intestinal cell kinase (ICK), a ubiquitously expressed and highly conserved serine/threonine protein kinase, supports cell proliferation. Results: Phosphorylation of Raptor Thr-908 by ICK is important for full activation of mTORC1. Conclusion: ICK plays an important role in regulating the mTORC1 activity. Significance: These findings provide a novel mechanistic insight into the signaling cascades by which ICK regulates proliferation.