Purification and characterization of two protein kinases acting on the aquaporin SoPIP2;1

AbstractAquaporins are water channel proteins that facilitate the movement of water and other small solutes across biological membranes. Plants usually have large aquaporin families, providing them with many ways to regulate the water transport. Some aquaporins are regulated post-translationally by phosphorylation. We have previously shown that the water channel activity of SoPIP2;1, an aquaporin in the plasma membrane of spinach leaves, was enhanced by phosphorylation at Ser115 and Ser274. These two serine residues are highly conserved in all plasma membrane aquaporins of the PIP2 subgroup. In this study we have purified and characterized two protein kinases phosphorylating Ser115 and Ser274 in SoPIP2;1. By anion exchange chromatography, the Ser115 kinase was purified from the soluble protein fraction isolated from spinach leaves. The Ca2+-dependent Ser274 kinase was purified by peptide affinity chromatography using plasma membranes isolated from spinach leaves. When characterized, the Ser115 kinase was Mg2+-dependent, Ca2+-independent and had a pH-optimum at 6.5. In accordance with previous studies using the oocyte expression system, site-directed mutagenesis and kinase and phosphatase inhibitors, the phosphorylation of Ser274, but not of Ser115, was increased in the presence of phosphatase inhibitors while kinase inhibitors decreased the phosphorylation of both Ser274 and Ser115. The molecular weight of the Ser274 kinase was approximately 50 kDa. The identification and characterization of these two protein kinases is an important step towards elucidating the signal transduction pathway for gating of the aquaporin SoPIP2;1

Plant Aquaporins: A study of expression, localization, specificity, and regulation

Aquaporins, or Major Intrinsic Proteins (MIPs), are integral proteins that facilitate transport of water and other small neutral solutes across biological membranes. They belong to a well conserved and ancient family of proteins, present in all organisms ranging from bacteria to plants and humans. The aquaporin family in plants is large, indicating complex and regulated water transport within the plant in order to adapt to different environmental conditions. All aquaporin isoforms probably work together in an orchestrated manner, where each individual aquaporin isoform displays a specific localization pattern, substrate specificity, and regulatory mechanism. When analyzing the whole genome of the model plant Arabidopsis thaliana, 35 aquaporin-encoded genes were identified. Based on sequence similarities and by phylogenetic analyses they were divided into four subfamilies; Plasma membrane Intrinsic Proteins (PIPs), Tonoplast Intrinsic Proteins (TIPs), NOD26-like Intrinsic Proteins (NIPs), and Small basic Intrinsic Proteins (SIPs). These subfamilies are conserved in many plant species. Based on the four subfamilies a new uniform nomenclature for all plant aquaporins was proposed, which is now widely accepted and used. A gene expression study, using a DNA microarray and quantitative real-time reverse transcriptase PCR, of all the 35 aquaporin genes in Arabidopsis was performed. The relative amounts of each isoform in leaves, roots, and flowers were analyzed, as well as their individual responses to drought stress. Focusing on four of the nine isoforms in the Arabidopsis NIP subfamily, i.e. AtNIP1;2, AtNIP2;1, AtNIP4;2, and AtNIP6;1, the gene expression on tissue and cell level was studied with promoter::GUS constructs, the protein localization was studied on the subcellular level in different organs with immunoblots, and the permeability to water and glycerol was examined by heterologous expression in Xenopus oocytes. The spinach leaf plasma membrane aquaporin SoPIP2;1 was heterologously overexpressed in the yeast Pichia pastoris, purified, and functionally characterized by reconstitution into proteoliposomes. The water channel activity of SoPIP2;1 has previously been shown to be regulated by phosphorylation of Ser115 and Ser274. Two protein kinases, acting on these two phosphorylation sites in SoPIP2;1, were partly purified and characterized

Initiation of biphasic insulin aspart 30/70 in subjects with type 2 diabetes mellitus in a largely primary care-based setting in Sweden.

AIMS: Despite a wealth of clinical trial data supporting use of the premixed insulin analogue, biphasic insulin aspart 30 (BIAsp 30) in the treatment of type 2 diabetes mellitus (T2DM), there is limited documentation of its use in primary care-based clinical practice. METHODS: An observational study investigating the safety and efficacy of BIAsp 30 in routine clinical practice was conducted. Patients were followed up 3 and 6 months after initiating insulin treatment. Safety and efficacy measures were documented. RESULTS: During the course of the study, 1154 patients were included (age range 20-95years), of whom 89% completed the 6-month follow-up period. Mean HbA(1c) at baseline was 8.8% (73mmol/mol), and had improved to 7.2% (55mmol/mol) after 6 months of treatment. The rate of total hypoglycaemia at completion of the study was 4.1 events per patient year. Major hypoglycaemic events were rare (two in total). CONCLUSIONS: BIAsp 30 was initiated safely and effectively in insulin-naïve patients with T2DM. The safety and efficacy profile observed in clinical trials was confirmed in this largely primary care-based setting in Sweden

Whole gene family expression and drought stress regulation of aquaporins

Since many aquaporins (AQPs) act as water channels, they are thought to play an important role in plant water relations. It is therefore of interest to study the expression patterns of AQP isoforms in order to further elucidate their involvement in plant water transport. We have monitored the expression patterns of all 35 Arabidopsis AQPs in leaves, roots and flowers by cDNA microarrays, specially designed for AQPs, and by quantitative real-time reverse transcriptase PCR (Q-RT-PCR). This showed that many AQPs are pre-dominantly expressed in either root or flower organs, whereas no AQP isoform seem to be leaf specific. Looking at the AQP subfamilies, most plasma membrane intrinsic proteins (PIPs) and some tonoplast intrinsic proteins (TIPs) have a high level of expression, while NOD26-like proteins (NIPs) are present at a much lower level. In addition, we show that PIP transcripts are generally down-regulated upon gradual drought stress in leaves, with the exception of AtPIP1;4 and AtPIP2;5, which are up-regulated. AtPIP2;6 and AtSIP1;1 are constitutively expressed and not significantly affected by the drought stress. The transcriptional down-regulation of PIP genes upon drought stress could also be observed on the protein level