Di-leucine Motif Of NOK/STYK1 Affects Its Protein Expression And Distribution In He La Cells

NOK/STYK1 is a structurally unique member in receptor tyrosine kinase family, which has strong ability to facilitate tumorigenesis and metastasis. It is also involved in cellular endocytosis. Analysis of NOK protein sequence revealed that there are several di-leucine motifs. Di-leucine motifs have been reported to play important roles in cellular endocytosis, vesicle trafficking, signaling sorting and et al. For this reason, in this study, we carried out the following two main points of work.Firstly, a series of mutants of NOK di-leucine motifs were established and their protein expression and cellular distribution pattern were analyzed in HeLa cells. The results indicated that mutation in different NOK di-leucine motifs caused different change of NOK protein expression level. The protein expression level of NOKL220 P mutant was higher than wild type NOK whereas NOKL41 P, NOKL203 P, NOKL237 P and NOKL299 P were lower than wild type. As to the cellular distribution, some mutants altered the cellular distribution pattern of NOK protein, including the proportion of dot pattern(DP)-containing cells to aggregation pattern(AP)-containing cells as well as the localization, the size and the number of NOK aggregates. The mutants of NOKL203 P, NOK237 P and L299 P significantly reduced the formation of NOK aggregates in cells. In addition to the above, overexpression of NOKL41 had more distinctive localization on nuclear envelop whereas overexpression of L220 P had more distinctive localization on cytoplasma membrane.Secondly, NOK gene were constructed to the different di-leucine motif point mutations vector about stable cell line, and verify their protein expression levels and changes in distribution in the cell characteristics. The results showed that, NOK and its di-leucine motif mutation carriers, there are some differences in the expression levels of stable cell lines, its trends and transiently transfected protein levels consistent. But in terms of positioning the cellular level, there are significant differences compared to stable cell lines and transient transfection. The stable cell lines mainly in locating cellular level distribution, significantly reduce the proportion of AP, and only in HeLa-NOKL41P-HA and HeLa-NOKL220P-HA appears in the AP distribution, and HeLa-NOK-HA, HeLa-NOKL203P-HA, HeLa-NOKL237P-HA, HeLa-NOKL299P-HA does not appear the AP distribution.Taken together, di-leucine motifs of NOK mediate its protein expression level and cellular distribution. Therefore, this study provides some new insights into understanding the molecular mechanism underlying the facilitation of NOK to tuomrigensis and metastasis.