Serine/threonine-kinase 33 (Stk33) - Component of the neuroendocrine network?

The present study was conducted to investigate the expression of serine/threonine-kinase 33 (Stk33) in neuronal structures of the central nervous system in rat and hamster as well as the presence of the protein in the brain of higher mammals, using a polyclonal antibody on cryosections of fixed brains. We found a distinct immunostaining pattern that included intense fluorescence of the ependymal lining of cerebral ventricles, and of hypothalamic tanycytes and their processes. We further observed intense staining of magnocellular neurons in the hypothalamic paraventricular, supraoptic and accessory neurosecretory nuclei, in particular the circular nuclei, and less intense stained neurons in other diencephalic regions. Double-immunostaining experiments showed a partial colocalization of Stk33 with arginine-vasopressin, oxytocin or neuronal nitric oxide-synthase in a large number of neurons of the hypothalamic nuclear regions. Colocalization of Stk33 with substance P or the catecholamine-synthesizing enzyme tyrosine-hydroxylase was not observed. Immunofluorescence was not found in autonomic regions of the lateral horn, suggesting that Stk33 does not contribute to hypothalamo-spinal connections. However, large Stk33-immunoreactive axonal projections from magnocellular hypothalamus to the neurohypophysis were evident. These functionally important connections provide the bridge from neuronal to humoral regulation of the endocrine system. Additionally, Western blots from mouse brain showed two distinct bands representing two Stk33 isoforms. We also present first evidence for the presence of Stk33/STK33 in neuronal structures, ependymal cells and tanycytes in tree shrew, baboon, and human brain.

Co-localization of serine/threonine kinase 33 (Stk33) and vimentin in the hypothalamus.

We investigate the immunoreactivity of serine/threonine kinase 33 (Stk33) and of vimentin in the brain of mouse, rat and hamster. Using a Stk33-specific polyclonal antibody, we show by immunofluorescence staining that Stk33 is present in a variety of brain regions. We found a strong staining in the ependymal lining of all cerebral ventricles and the central canal of the spinal cord as well as in hypothalamic tanycytes. Stk33 immunoreactivity was also found in circumventricular organs such as the area postrema, subfornical organ and pituitary and pineal glands. Double-immunostaining experiments with antibodies against Stk33 and vimentin showed a striking colocalization of Stk33 and vimentin. As shown previously, Stk33 phosphorylates recombinant vimentin in vitro. Co-immunoprecipitation experiments and co-sedimentation assays indicate that Stk33 and vimentin are associated in vivo and that this association does not depend on further interacting partners (Brauksiepe et al. in BMC Biochem 9:25, 2008). This indicates that Stk33 is involved in the dynamics of vimentin polymerization/depolymerization. Since in tanycytes the vimentin expression is regulated by the photoperiod (Kameda et al. in Cell Tissue Res 314:251-262, 2003), we determine whether this also holds true for Stk33. We study hypothalamic sections from adult Djungarian hamsters (Phodopus sungorus) held under either long photoperiods (L:D 16:8 h) or short photoperiods (L:D 8:16 h) for 2 months. In addition, we examine whether age-dependent changes in Stk33 protein content exist. Our results show that Stk33 in tanycytes is regulated by the photoperiod as is the case for vimentin. Stk33 may participate in photoperiodic regulation of the endocrine system.

Evaluation of the hormonal state of columnar apple trees (Malus x domestica) based on high throughput gene expression studies.

The columnar phenotype of apple trees (Malus x domestica) is characterized by a compact growth habit with fruit spurs instead of lateral branches. These properties provide significant economic advantages by enabling high density plantings. The columnar growth results from the presence of a dominant allele of the gene Columnar (Co) located on chromosome 10 which can appear in a heterozygous (Co/co) or homozygous (Co/Co) state. Although two deep sequencing approaches could shed some light on the transcriptome of columnar shoot apical meristems (SAMs), the molecular mechanisms of columnar growth are not yet elaborated. Since the influence of phytohormones is believed to have a pivotal role in the establishment of the phenotype, we performed RNA-Seq experiments to study genes associated with hormone homeostasis and clearly affected by the presence of Co. Our results provide a molecular explanation for earlier findings on the hormonal state of columnar apple trees. Additionally, they allow hypotheses on how the columnar phenotype might develop. Furthermore, we show a statistically approved enrichment of differentially regulated genes on chromosome 10 in the course of validating RNA-Seq results using additional gene expression studies.

The Serine/threonine kinase Stk33 exhibits autophosphorylation and phosphorylates the intermediate filament protein Vimentin.

BACKGROUND: Colocalization of Stk33 with vimentin by double immunofluorescence in certain cells indicated that vimentin might be a target for phosphorylation by the novel kinase Stk33. We therefore tested in vitro the ability of Stk33 to phosphorylate recombinant full length vimentin and amino-terminal truncated versions thereof. In order to prove that Stk33 and vimentin are also in vivo associated proteins co-immunoprecipitation experiments were carried out. For testing the enzymatic activity of immunoprecipitated Stk33 we incubated precipitated Stk33 with recombinant vimentin proteins. To investigate whether Stk33 binds directly to vimentin, an in vitro co-sedimentation assay was performed. RESULTS: The results of the kinase assays demonstrate that Stk33 is able to specifically phosphorylate the non-alpha-helical amino-terminal domain of vimentin in vitro. Furthermore, co-immunoprecipitation experiments employing cultured cell extracts indicate that Stk33 and vimentin are associated in vivo. Immunoprecipitated Stk33 has enzymatic activity as shown by successful phosphorylation of recombinant vimentin proteins. The results of the co-sedimentation assay suggest that vimentin binds directly to Stk33 and that no additional protein mediates the association. CONCLUSION: We hypothesize that Stk33 is involved in the in vivo dynamics of the intermediate filament cytoskeleton by phosphorylating vimentin.

Differential expression pattern of the novel serine/threonine kinase, STK33, in mice and men.

Serine/threonine kinase 33 (STK33/Stk33) is a recently discovered gene whose inferred amino acid sequence translation displays characters typical for a calcium/calmodulin dependent kinase (CAMK). In this study we analysed the STK33/Stk33 RNA and protein distribution and the localization of the protein. The STK33/Stk33 expression pattern resembles those of some related members of the CAMK group. STK33/Stk33 displays a nonubiquitous and, in most tissues, low level of expression. It is highly expressed in testis, particularly in cells from the spermatogenic epithelia. Moreover, significant expression is detected in lung epithelia, alveolar macrophages, horizontal cells in the retina and in embryonic organs such as heart, brain and spinal cord. A possible role of STK33/Stk33 in spermatogenesis and organ ontogenesis is discussed.