We report here a novel ZIP kinase isoform (hZIPK-S) in which a majority of originally assigned exon 8, encoding the C-terminal non-kinase domain, is spliced out. This type of splicing has been known as intron retention [31
]. A similar type of the splicing variants are found for myosin IXb [33
](Kambara and Ikebe, unpublished observation). Previously, MacDonald et al. [20
] reported that ZIP-like kinase purified from cow bladder had an apparent molecular mass of 32 kDa that was much smaller than the reported mass of ZIP kinase, although one cannot eliminate the possibility that the 32 kDa peptide is produced by proteolytic degradation of ZIP Kinase during biochemical purification from cow bladder tissues. The calculated molecular mass of hZIPK-S found in the present study is 37kDa, and an apparent molecular mass estimated with the mobility of SDS-PAGE is 34 kDa. Based upon the similarity of the molecular mass, previously reported ZIP-like kinase may be the short isoform of human ZIP kinase (hZIPK-S) found in the present study. However, the partial amino acid sequence of ZIP-like kinase determined by Edman degradation of the three isolated peptides showed that seven amino acid residues out of the thirty four determined residues were different from those of kinase domain of ZIP kinase. On the other hand, the kinase domain of hZIPK-S was completely identical to that of the long isoform (hZIPK-L). The amino acid sequence of the kinase domain of ZIP kinase is highly conserved among the mammalian species [1
] and the region of the three peptides, determined for ZIP-like kinase, is completely identical among the ZIP kinase from various species. Therefore, it is unlikely that the different sequence of bovine ZIP-like kinase from ZIP kinases is due to the species difference. Therefore, ZIP-like kinase might be different from the short isoform of ZIP kinase (hZIPK-S) identified in the present study, although one cannot eliminate the possibility that the difference in the sequence is due to the amino acid sequencing error of the ZIP-like kinase.
It was reported recently that there were no spliced variants of the ZIP kinase in vascular smooth muscle [34
]. Our result clearly demonstrated that there is a short isoform present in smooth muscle tissues that is produced due to the splicing at the exon 8. As shown in , the original 3′ untranslated region is almost completely deleted in hZIPK-S and the presence of this isoform could only be found using the 3′ RACE technique. The sequence used for the minus strand PCR primers in the previous study are at the regions upstream of the 5′ region of the exon 8 and these primers do not yield the PCR product containing hZIPK-S unique sequence. Therefore, it is reasonable to assume that the previous study could not detect the present short ZIP kinase isoform (ZIPK-S)[34
We found that hZIPK-S can bind to MYPT1 similar to hZIPK-L. The result indicates that the C-terminal domain of the long isoform including the leucine zipper is not critical to the binding of ZIP kinase to MYPT1. We found that the kinase domain of hZIPK (Met1-Ala277, hZIPK-KD) has no binding activity to MYPT1, suggesting that the amino acids, Ile278-Ser312, at least in part responsible for the MYPT1 binding site of ZIP kinase. On the other hand, we found that both the hZIPK-S and hZIPK-KD bind to myosin. This result suggests that the myosin binding site is different from that of the MYPT1 binding site.
Initially nuclear localization of rodent ZIP kinase was reported in association with cell death inducing activity [3
]. Subsequently, human ZIP kinase was cloned from Hela cells [5
], which also induces cell death in Hela cells and showed essentially cytoplasmic localization. Our data supports that, unlike rodent ZIP kinases, human ZIP kinase resides in cytoplasm and may play a role other than inducing cell death in the cytoplasm. Interestingly, the kinase activity of the ZIPK-S was significantly higher for myosin but not for MYPT1. Furthermore, our biochemical analysis revealed that the ZIPK-S has different kinase properties from the ZIPK-L, suggesting that the C-terminal domain including the leucine zipper of ZIP kinase is important for its substrate specificity. Consistently, it was reported that the leucine zipper of human ZIP kinase (ZIPK-L) affects its enzymatic activity and cellular localization [30
It was previously shown that ZIP kinase localizes at the stress fiber in mammalian motile cells where a significant level of di-phosphorylated MLC20
is localized (18). In the present study, we found the localization of the newly found hZIPK-S isoform at the stress fiber in addition to the previously found hZIPK-L isoform. Furthermore, we found that hZIPK isoforms were co-localized with myosin at the stress fiber. These results are consistent with the finding that hZIPK isoforms bind to myosin in vitro. Based on these findings, it is thought that hZIPK binds to myosin at the stress fibers where hZIPK isoforms phosphorylates myosin to stabilize the thick filament formation. On the other hand, it has been shown that MYPT1 in part localized at the stress fiber [35
]. Therefore, it is plausible that hZIPK also phosporylates the inhibitory site of MYPT1 at the stress fiber thus down-regulating the MLCP activity, and attenuates the dephosphorylation of myosin II at the stress fiber.
It was shown previously that ZIP kinase undergoes autophosphorylation, yet the effect of autophosphorylation on the function has been obscure. We examined the effect of autophosphorylation on hZIPK-S function. Of interest is whether the autophosphorylation of hZIPK-S alters the binding to MYPT1 because the region overlaps the segment responsible for the binding of hZIPK-S to MYPT1. However, we could not find a significant effect of autophosphorylation on the binding to MYPT1. On the other hand, we found a significant decrease in the binding of hZIPK-S to the targeting proteins, i.e., myosin and MYPT1, when they were phosphorylated. The result suggests that ZIP kinase leaves away from the targeting proteins as soon as it finishes phosphorylation. Therefore, it is thought that the localization of hZIPK isoforms at the stress fiber predominantly reflects the binding to the unphosphorylated myosin present in the stress fiber. Because it is reasonable to assume that the number of the ZIP kinase molecules is much less than those of myosin II and MYPT1, the low affinity of ZIP kinase for the phosphorylated target proteins enables it to efficiently phopshorylate the unphosphorylated target proteins.
Our results indicate that the major phosphorylation sites of ZIP kinase are centered within Ile278-Ser312, which is included in the short isoform found in the present study, but not in the catalytic core. Graves et al. [30
] reported that human ZIP kinase expressed in HEK293 cells is autophosphorylated at Thr180 and Thr225, and Thr265 in the catalytic core revealed by 32
P incorporation into the corresponding peptides. They claimed that the phosphorylation was necessary for the kinase activity of ZIP kinase because the mutation at Thr180, Thr225, or Thr265 abolished the kinase activity. The apparent discrepancy of the results is unclear. However, since the reported three residues are in the kinase activation T loop, the substrate binding groove, and the kinase subdomain X, respectively [30
], it is likely that the mutation itself disturbs the structure of the kinase thus inactivating the enzyme activity. Supporting this notion, T180D and T225D mutation caused nearly complete inactivation of the kinase rather than mimicking phosphorylation (activation) [30
]. Interestingly, the kinase dead ZIP kinase was also phosphorylated at S311 and T265 that was argued to be phosphorylated by endogenous ZIP kinase associated with the dead kinase [30
], therefore, the observed 32
P incorporation to Thr180, Thr225 and Thr265 might be catalyzed by the associated kinases.
Quite recently, it was shown that the kinase domain of the recombinant mouse ZIP kinase expressed in E. coli
can be autophosphorylated at Thr265 and this plays an important role in its activity [36
]. The apparent discrepancy from our data is likely due to the difference in the expression systems. As is known, many protein kinases are phosphorylated right after the protein synthesis which is important for the activation of the kinase activity in eukaryote, while such an apparatus is lacking in prokaryote. Therefore, it is reasonable to assume that the expressed huZIPK in sf9 cells is pre-phosphorylated by endogenous kinases due to post-translational modification. Supporting this notion, it is shown that ZIP kinase can be phosphorylated by endogenous kinases associated with ZIP kinase in mammalian cultured cells [30
]. These results suggest that ZIP kinase is phosphorylated at the activation loop by post translational modification thus producing an active kinase and the additional sites located at the C-terminal side of the catalytic domain are phosphorylated either by autophosphorylation or by other kinases. At present, the significance of the regulatory phosphorylation on the function of ZIP kinase is obscure and the understanding of the regulation of ZIP kinase requires further study.