Nuclear Localization Signal of Murine CMP-Neu5Ac Synthetase Includes Residues Required for Both Nuclear Targeting and Enzymatic Activity

Münster, Anja-K.; Weinhold, Birgit; Gotza, Birgit; Mühlenhoff, Martina; Frosch, Matthias; Gerardy‐Schahn, Rita

doi:10.1074/jbc.m201093200

Cited by 36 publications

(57 citation statements)

References 55 publications

(48 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…14 While hIK1 possesses a cytoplasmic NH 2 -terminal multi-basic motif (RRRKR) closely apposed to the predicted first transmembrane domain, 30,31 the role of this domain has not previously been evaluated. Multi-basic motifs are known to play crucial roles in a wide array of cell biological processes, including ER exit, 17,32 ER retention, 15,16 acting as nuclear localization signals 33,34 as well as the gating of K + channels. 18,19 Thus, the role these multi-basic motifs play in protein trafficking and function is dependent upon the context in which they are expressed.…”

Section: Discussionmentioning

confidence: 99%

An NH2-Terminal Multi-Basic RKR Motif Is Required for the ATP-Dependent Regulation of hIK1

et al. 2007

View full text Add to dashboard Cite

We previously demonstrated that the ATP/PKA-dependent activation of the human intermediate conductance, Ca 2+ -activated K + channel, hIK1, is dependent upon a C-terminal motif. The NH 2 -terminus of hIK1 contains a multi-basic 13 RRRKR 17 motif, known to be important in the trafficking and function of ion channels. While individual mutations within this domain have no effect on channel function, the triple mutation ( 15 RKR 17 /AAA), as well as additional double mutations, result in a near complete loss of functional channels, as assessed by whole-cell patch-clamp. However, cell-surface immunoprecipitation studies confirmed expression of these mutated channels at the plasma membrane. To elucidate the functional consequences of the 15 RKR 17 /AAA mutation we performed inside-out patch clamp recordings where we observed no difference in Ca 2+ affinity between the wild-type and mutated channels. However, in contrast to wild-type hIK1, channels expressing the 15 RKR 17 /AAA mutation exhibited rundown, which could not be reversed by the addition of ATP. Wild-type hIK1 channel activity was reduced by alkaline phosphatase both in the presence and absence of ATP, indicative of a phosphorylation event, whereas the 15 RKR 17 /AAA mutation eliminated this effect of alkaline phosphatase. Further, single channel analysis demonstrated that the 15 RKR 17 /AAA mutation resulted in a four-fold lower channel open probability (P o ), in the presence of saturating Ca 2+ and ATP, compared to wild-type hIK1. In conclusion, these results represent the first demonstration for a role of the NH 2 -terminus in the second messenger-dependent regulation of hIK1 and, in combination with our previous findings, suggest that this regulation is dependent upon a close NH 2 /C-terminal association.

show abstract

Section: Discussionmentioning

confidence: 99%

An NH2-Terminal Multi-Basic RKR Motif Is Required for the ATP-Dependent Regulation of hIK1

et al. 2007

View full text Add to dashboard Cite

show abstract

“…1A). P-3F ax -Neu5Ac presumably enters the intracellular sialic acid synthesis pathway via a salvage pathway and is converted to CMP-P-3F ax -Neu5Ac (23). Only CMP-activated sialic acids can be recognized by the CMP-sialic acid transporter and undergo active transportation into the Golgi lumen where they serve as substrate for sialyltransferases (24,25).…”

Section: Introductionmentioning

confidence: 99%

Targeting Aberrant Sialylation in Cancer Cells Using a Fluorinated Sialic Acid Analog Impairs Adhesion, Migration, and In Vivo Tumor Growth

Büll

Boltje

Wassink

et al. 2013

Molecular Cancer Therapeutics

150

133

View full text Add to dashboard Cite

Cancer cells decorate their surface with a dense layer of sialylated glycans by upregulating the expression of sialyltransferases and other glycogenes. Although sialic acids play a vital role in many biologic processes, hypersialylation in particular has been shown to contribute to cancer cell progression and metastasis. Accordingly, selective strategies to interfere with sialic acid synthesis might offer a powerful approach in cancer therapy. In the present study, we assessed the potential of a recently developed fluorinated sialic acid analogue (P-3F ax -Neu5Ac) to block the synthesis of sialoglycans in murine melanoma cells and the consequences on cell adhesion, migration, and in vivo growth. The results showed that P-3F ax -Neu5Ac readily caused depletion of a2,3-/a2,6-linked sialic acids in B16F10 cells for several days. Long-term inhibition of sialylation for 28 days was feasible without affecting cell viability or proliferation. Moreover, P-3F ax -Neu5Ac proved to be a highly potent inhibitor of sialylation even at high concentrations of competing sialyltransferase substrates. P-3F ax -Neu5Ac-treated cancer cells exhibited impaired binding to poly-L-lysine, type I collagen, and fibronectin and diminished migratory capacity. Finally, blocking sialylation of B16F10 tumor cells with this novel sialic acid analogue reduced their growth in vivo. These results indicate that P-3F ax -Neu5Ac is a powerful glycomimetic capable of inhibiting aberrant sialylation that can potentially be used for anticancer therapy. Mol Cancer Ther; 12(10); 1935-46. Ó2013 AACR.

show abstract

“…A unique feature of the eukaryotic sialylation pathway (Supplemental Figure 1) is CMAS' nuclear sequestration, 10,11 which is dispensable for functionality at the cellular level. 12 In this study, we describe a mouse model with mutant Cmas. Using a knock-in approach, the major nuclear localization signal was inactivated by point mutations (nls).…”

mentioning

confidence: 99%

“…RPAR) clearly relocated to the cytoplasm of transfected cells but retained full enzymatic activity. 12 To test the consequences of CMAS relocation in a mouse model, exon 4 of the endogenous Cmas was replaced by Cmas nls exon 4 ( Figure 1A). The neomycin resistance gene was removed by crossing with the deleter strain Zp3-cre.…”

mentioning

confidence: 99%

Deficits in Sialylation Impair Podocyte Maturation

Weinhold

Sellmeier

Schaper

et al. 2012

Journal of the American Society of Nephrology

Self Cite

View full text Add to dashboard Cite

The role of sialylation in kidney biology is not fully understood. The synthesis of sialoglycoconjugates, which form the outermost structures of animal cells, requires CMP-sialic acid, which is a product of the nuclear enzyme CMAS. We used a knock-in strategy to create a mouse with point mutations in the canonical nuclear localization signal of CMAS, which relocated the enzyme to the cytoplasm of transfected cells without affecting its activity. Although insufficient to prevent nuclear entry in mice, the mutation led to a drastically reduced concentration of nuclear-expressed enzyme. Mice homozygous for the mutation died from kidney failure within 72 hours after birth. The Cmas nls mouse exhibited podocyte foot process effacement, absence of slit diaphragms, and massive proteinuria, recapitulating features of nephrinknockout mice and of patients with Finnish-type congenital nephrotic syndrome. Although the Cmas nls mouse displayed normal sialylation in all organs including kidney, a critical shortage of CMP-sialic acid prevented sialylation of nephrin and podocalyxin in the maturing podocyte where it is required during the formation of foot processes. Accordingly, the sialylation defects progressed with time and paralleled the morphologic changes. In summary, sialylation is critical during the development of the glomerular filtration barrier and required for the proper function of nephrin. Whether altered sialylation impairs nephrin function in human disease requires further study.

show abstract

Nuclear Localization Signal of Murine CMP-Neu5Ac Synthetase Includes Residues Required for Both Nuclear Targeting and Enzymatic Activity

Cited by 36 publications

References 55 publications

An NH2-Terminal Multi-Basic RKR Motif Is Required for the ATP-Dependent Regulation of hIK1

An NH2-Terminal Multi-Basic RKR Motif Is Required for the ATP-Dependent Regulation of hIK1

Targeting Aberrant Sialylation in Cancer Cells Using a Fluorinated Sialic Acid Analog Impairs Adhesion, Migration, and In Vivo Tumor Growth

Deficits in Sialylation Impair Podocyte Maturation

Contact Info

Product

Resources

About