A non-catalytic role for inositol 1,3,4,5,6-pentakisphosphate 2-kinase in the synthesis of ribosomal RNA

Abstract

SummaryFundamental to the life and destiny of every cell is the regulation of protein synthesis through ribosome biogenesis, which begins in the nucleolus with the production of ribosomal RNA (rRNA). Nucleolar organization is a highly dynamic and tightly regulated process; the structural factors that direct nucleolar assembly and disassembly are just as important in controlling rRNA synthesis as are the catalytic activities that synthesize rRNA. Here, we report that a signaling enzyme, inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP5K) is also a structural component in the nucleolus. We demonstrate that IP5K has functionally significant interactions with three proteins that regulate rRNA synthesis: protein kinase CK2, TCOF1 and upstream-binding-factor (UBF). Through molecular modeling and mutagenic studies, we identified an Arg-Lys-Lys tripeptide located on the surface of IP5K that mediates its association with UBF. Nucleolar IP5K spatial dynamics were sensitive to experimental procedures (serum starvation or addition of actinomycin D) that inhibited rRNA production. We show that IP5K makes stoichiometrically sensitive contributions to the architecture of the nucleoli in intact cells, thereby influencing the degree of rRNA synthesis. Our study adds significantly to the biological significance of IP5K; previously, it was the kinase activity of this protein that had attracted attention. Our demonstration that IP5K 'moonlights' as a molecular scaffold offers an unexpected new example of how the biological sophistication of higher organisms can arise from gene products acquiring multiple functions, rather than by an increase in gene number.

Inositol pyrophosphates promote tumor growth and metastasis by antagonizing liver kinase B1

“…2F) and the percentage of metastases to liver (Fig. 2G), the most common site of metastasis for The inositol polyphosphate kinases, including IP6Ks, have both catalytic activity-dependent and -independent functions (1,5,11,27,28). To examine whether the oncogenic actions of IP6K2 depend on its catalytic activity, we established rescue cell lines by retroviral expression of wild-type (WT-R) or kinase-dead K222A IP6K2 (29) (KD-R) in IP6K2 −/− cells.…”

Crystallization and Preliminary X-Ray Diffraction Analysis of a Mammal Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase

“…A reported study suggested that human IP 5 2-K presents a different putative Zinc binding motif possibly related with the enzyme capacity to translocate to the nucleus [2]. Moreover, mIP 5 2-K associates with several proteins involved in rRNA synthesis; but data of possible interacting regions are lacking [9]. Considering that the ipk1 gene deletion in plants does not present a dramatic effect while in mammals the embryo is not viable [30], this enzyme seems to present specific features and roles in mammals.…”

“…Moreover IP 6 is the precursor of pyrophosphate inositol diphospho-inositol pentakisphosphate (IP 7 ) and bisdiphosphoinositol tetrakisphosphate (IP 8 ) [6], which are involved in apoptosis processes [7,8]. Very recently, IP 5 2-K has been shown to present a double functionality; apart from its kinase function it presents a structural role in the cell nucleolus, interacting with at least three proteins involved in the regulation of rRNA synthesis [9]. In concordance with the important functions ascribed to IP 5 2-K in mammals, disruption of its gene produces early lethality and non-viable murine embryos [10].…”

Synthesis of Biotinylated Inositol Hexakisphosphate To Study DNA Double-Strand Break Repair and Affinity Capture of IP6-Binding Proteins

“…While nucleolin has not previously been identified as an IP-binding protein, co-localization of nucleolin with IPK1, the primary enzyme that produces IP 6 through phosphorylation of IP 5 , and phosphorylation of nucleolin by IP 7 link nucleolin with IP 6 metabolism and suggest that nucleolin may interact with IP 6 (15,(53)(54)(55). Western blot analysis using anti-nucleolin antibodies identified nucleolin as 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 11 one of the proteins retained by IP 6 -biotin affinity capture ( Figure 4B).…”

“…We used a candidate protein approach to determine whether a likely IP 6 -binding protein was affinity captured by IP 6 -biotin. Nucleolar localization of the IP 5 kinase that produces IP 6 suggests a role for IP 6 in the nucleolus (15,53). When expressed in yeast, the human nucleolar protein nucleolin can be directly phosphorylated by the inositol pyrophosphate IP 7 (see supplemental figure S3B, (55)), which suggests a possible physical interaction with highly phosphorylated IPs such as IP 6 (54,55).…”