Isolation and functional characterization of the C-terminus of rice phosphatidylinositol 4-kinase in vitro

Abstract

A partial rice (Oryza sativa L.) cDNA clone, OsPI4Klc, was isolated through screening of a cDNA library constructed from tillering materials. OsPI4Klc encoded a peptide of 608 amino acids with a calculated molecular mass of 68.4 kDa. The OsPI4Klc peptide shared high homology and possessed the highly conserved domains present in most isolated cloned P14-kinases, i.e. a lipid kinase unique (LKU) domain and a catalytic (CAT) domain. A region with similarity to pleckstrin homology (PH) domain was present in OsPI4Klc as well. Further comparison with genomic sequences in databases revealed that OsPI4Klc is located at the 3 end of a putative rice PI 4-kinase coding gene OsPI4K1, and its coding region corresponded to the C-terminal half of OsPI4K1 protein. Twelve exons (49-562 bp in size) and 11 introns (77-974 bp in size) were identified in OsPI4K1c. The recombinant protein expressed in Escherichia coli phosphorylates phosphatidylinositol at the D4 position of the inositol ring. OsPI4K1 transcript levels were detected in a low but constitutive manner in shoot, stem, leaf, spike and root tissues and did not change upon treatment with different hormones, calcium and jasmonic acid (JA). However, treatment with salicylic acid (SA) elevated the mRNA level of the OsPI4K1 gene, which suggested the involvement of OsPI4Kl in wounding responses.

DNA chip-based expression profile analysis indicates involvement of the phosphatidylinositol signaling pathway in multiple plant responses to hormone and abiotic treatments

“…Analysis on A. thaliana genome identified four members of the family (two known, two putative) and studies via DNA chips indicated that expression of PI4Kα2 was relatively high in leaves though it did not change following hormone treatment or stress, while PI4Kβ1 and PI4Kβ2 were present in the normal tissues and up-regulated by auxin and down-regulated by cytokinin, respectively. Additionally, expressions of both were regulated following SA treatment and cold stress (Tab 3), in a manner similar to that seen in rice OsPI4K1 [8], which was induced by SA, suggesting the involvement of PI4K in plant wounding responses.…”

“…In a tightly regulated manner, these second messengers may be recycled by further phosphorylation of Ins(1, 4, 5)P 3 to Ins(1, 3, 4, 5)P 4 by Ins (1, 4, 5) P 3 3-kinase (IP3K), or dephosphorylation to Ins(1, 4)P 2 by inositol polyphosphate 5-phosphatase (IPPase). To date, most molecular characterizations, expression pattern analyses and biochemical studies have focused on key PI pathway-related proteins including PIS [5], PI4K [6][7][8], PIP5K [9][10][11][12][13], PLC [14,15], PLD [16], PLA 1 [17], PLA 2 [18,19], PI 3-kinase (PI3K) [20], IPPase [21] and inositol 1, 3, 4-trisphosphate 5/6-kinase (IP 3 5/6K) [22]. In contrast, little attention has been paid to other proteins such as inositol 1, 4, 5-trisphosphate receptor (IP 3 R) and PI transfer protein (PITP), and there are many other signal molecules yet to be investigated within this pathway.…”

The Highly Charged Region of Plant β-type Phosphatidylinositol 4-kinase is Involved in Membrane Targeting and Phospholipid Binding

“…Additionally, besides the membrane binding, the PH domain of plant a-type PI4K is also involved in lipids binding, although the catalytic domain of OsPI4K1, a a-type PI4K, could phosphorylate PI4P in the absence of the PH domain (Kong et al, 2003). Our results demonstrate that the PPC region of b-type PI4Ks is involved in phospholipid binding, i.e.…”

“…The previously identified OsPI4K1 gene encodes a a-type PI4K (Kong et al, 2003) and the second one, named OsPI4K2 here (GenBank Acc. No.…”

“…In plant cells, PI4K activity is present in many compartments, including the plasma membrane (Kamada and Muto, 1991;Gross et al, 1992), cytosol (Okpodu et al, 1995), and cytoskeleton (Xu et al, 1992). In rice, OsPI4K1 was found to be constitutively expressed in various tissues at a low level and was up-regulated by salicylic acid, suggesting the involvement of OsPI4K1 in wounding responses (Kong et al, 2003).…”