Characterization and Partial Purification of Microsomal Casein Kinase II from Osteoblast-like Cells: An enzyme that phosphorylates osteopontin and phosphophoryn

Wu, Chuansha; Shimizu, Yushiyuki; Ng, A. M. L.; Pan, Yu-Min

doi:10.3109/03008209609028890

Cited by 6 publications

(6 citation statements)

References 43 publications

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“…d Lack of effect of 2 mM Pi and 5 ng/ml TNF-α treatments on Pit-1 abundance and distribution. Size bars, 20 µm kinases involved in the phosphorylation of secreted matrix proteins like ostepontin [34]. Therefore, when vascular calcification is prevented by knocking-down the expression of Pit-1 with RNA interference [14], it is now unclear whether the effect is based on the impairment of Pi transport in the cell membrane, or the impairment of an unknown function in the endoplasmic reticulum.…”

Section: Discussionmentioning

confidence: 98%

Vascular smooth muscle cell calcification and SLC20 inorganic phosphate transporters: effects of PDGF, TNF-α, and Pi

Villa‐Bellosta

Levi

Sorribas

2009

Pflugers Arch - Eur J Physiol

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Pi transport by vascular smooth muscle cells (VSMC) has been proposed to play an important role in the pathogenesis of vascular calcification. In this study, we have determined the correlation between calcification induced by Pi, platelet-derived growth factor (PDGF)-BB, and tumor necrosis factor-alpha and Pi transport activity in primary cultures of rat aortic VSMC. These agents induced calcification and increased the expression of Cbfa1, Msx2, and Bmp2 osteogene messenger RNA in rat aortic VSMC, while Pi transport rate was not modified per milligram of protein. Only PDGF increased Pi transport when it was expressed per unit of DNA, as PDGF also increased total cell protein by 100%, while DNA content and number of cells were not modified. PDGF increased the expression of the Pi transporter, Pit-1, but membrane protein biotinylation showed that Pit-1 abundance was not modified in the cell surface. Immunofluorescence revealed that, under basal conditions, Pit-1 is only slightly expressed at the cell membrane, but strongly expressed inside the cell. The intracellular signal colocalizes with endoplasmic reticulum (ER) markers, and PDGF increases Pit-1 expression in the ER but not the cell membrane. In conclusion, Pi transport across the plasma membrane does not correlate directly with calcification, but the expression of Pit-1 in the ER opens new possibilities for the study of the pathogenesis of vascular calcification.

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Section: Discussionmentioning

confidence: 98%

Vascular smooth muscle cell calcification and SLC20 inorganic phosphate transporters: effects of PDGF, TNF-α, and Pi

Villa‐Bellosta

Levi

Sorribas

2009

Pflugers Arch - Eur J Physiol

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“…) bone or dentin ECM molecules which remained associated with the ECM surrounding the nodules. In the case of odontoblast culture The work from our laboratories (Wu et al, 1992(Wu et al, , 1995(Wu et al, , 1996Sfeir andVeis, 1995, 1996;Sfeir, 1996) (Sfeir andVeis, 1995, 1996;Wu et al, 1995Wu et al, , 1996 Figure 9. Evidence for CK2o--subunit isoforms in ROS 17/2.8 cells.…”

Section: (C) Other Bone Ncpsmentioning

confidence: 99%

“…Higher centrifugal forces over a longer period of time (34,000 x g, 30 min) then allow for the separation of the Golgi and heavy microsomes from the cytosolic components. The membrane-associated proteins can be obtained from detergent-solubilized microsomal fractions, centrifuged again at high speed (Wu et al, 1995(Wu et al, , 1996. Any one of the fractions can then be studied, depending on one's purpose.…”

Section: (C) Other Bone Ncpsmentioning

confidence: 99%

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Phosphorylation of the Proteins of the Extracellular Matrix of Mineralized Tissues By Casein Kinase-Like Activity

Veis

Sfeir

1997

Critical Reviews in Oral Biology & Medicine

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The extracellular matrix of the connective tissue contains non-collagenous proteins (NCP) which are acidic in character. The NCP of mineralizing systems (bone, dentin) differ from those of the non-mineralizing systems (skin, tendon) in that the mineralized tissue NCP are frequently phosphorylated. The phosphorylated proteins have been implicated in various aspects of the mineralization process. Thus, it is of interest to consider the mechanism and regulation of phosphorylation of the major matrix NCP. The majority of the phosphorylation takes place at Ser or Thr residues embedded within acidic sequences, and therefore are targets for casein kinase I (CKI ) or casein kinase 11 (CK2)-like kinases. CKI and CK2 are distantly related members of the protein kinase family. They are ubiquitous, constitutively active, second-messenger-independent kinases. CKI is found in a variety of isoforms, all homologous to the cx-subunit of the protein kinase family. It acts as a monomer. The active form of CK2 is a tetrameric holoenzyme, with 2 cx catalytic subunits and 2 ( regulatory subunits. The CK2 ac has activity alone, but the holoenzyme is four-to five-fold that activity. CK2 can use either ATP or GTP as the phosphate donor, but CK 1 can use only ATP. The CK2 activity which phosphorylates the mineralized tissue NCP appears to be localized to membraneassociated cell fractions, and is present in the endoplasmic reticulum and Golgi compartments in osteoblasts, where phosphorylation of the secreted proteins appears to take place as co-and post-translational processes. Data Bone and dentin are tissues in which mineral deposits of carbonate apatite form in an orderly fashion within a pre-formed collagen fibril matrix. Structural studies clearly show that the majority of the mineral phase is composed of carbonate-apatite crystals, whose crystal c-axes are aligned with the fibril axes of the collagen fibrils (FittonJackson, 1957;Glimcher and Krane, 1968;Glimcher, 1976;Weiner and Traub, 1986;Arsenault, 1988;Arsenault et al, 1991;Landis et al., 1992). Although much of the early work attempted to show that the collagen fibrils were responsible for the placement and induction of mineral deposition, this proposition was never verified. Instead, attention began to focus on the non-collagenous protein components of the matrix (Herring and Kent, 1963;Schlueter and Veis, 1964;Veis and Schlueter, 1964;Spector and Glimcher, 1972;Veis et al., 1972;Glimcher, 1976), especially since it was recognized that, as a class, the non-collagenous extracellular proteins (NCP) shared a common char360Crit Rev Oral Biol Med 8(4)

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“…Specifically, we focus on a unique extracellular matrix protein, phosphophoryn, which undergoes a high degree of phosphorylation during its synthesis. As described, our vector design directs the recombinant PP first to the endoplasmic reticulum (ER), subsequently channeling the protein through the secretory pathway (ER, Golgi, and secretory vesicles) where it undergoes post-translational modification (26,27,29,30).…”

Section: Discussionmentioning

confidence: 99%

Expression of Phosphophoryn Is Sufficient for the Induction of Matrix Mineralization by Mammalian Cells

Sfeir

Lee

et al. 2011

Journal of Biological Chemistry

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Mineralized tissues such as dentin and bone assemble extracellular matrices uniquely rich in a variety of acidic phosphoproteins. Although these proteins are presumed to play a role in the process of biomineralization, key questions regarding the nature of their contributions remain unanswered. First, it is not known whether highly phosphorylated proteins alone can induce matrix mineralization, or whether this activity requires the involvement of other bone/dentin non-collagenous proteins. Second, it remains to be established whether the protein kinases that phosphorylate these acidic proteins are unique to cells responsible for producing mineralized tissues. To begin to address these questions, we consider the case of phosphophoryn (PP), due to its high content of phosphate, high affinity for Ca 2؉ , and its potential role in hydroxyapatite nucleation. We have created a model system of biomineralization in a cellular environment by expressing PP in NIH3T3 fibroblasts (which do not produce a mineralized matrix); as a positive control, PP was expressed in MC3T3-E1 osteoblastic cells, which normally mineralize their matrices. We show that expression of PP in NIH3T3 cells is sufficient for the induction of matrix mineralization. In addition, assessment of the phosphorylation status of PP in these cells reveals that the transfected NIH3T3 cells are able to phosphorylate PP. We suggest that the phosphorylation of PP is essential for mineral formation. The principle goal of this study is to enrich the current knowledge of mineralized tissue phosphorylation events by analyzing them in the context of a complete cellular environment.In biologically induced mineralization, crystals are generally produced by a heterogeneous nucleation mechanism. The deposition of mineral crystals in bone, dentin, and cartilage is orchestrated by cells, and the growth of these crystals is facilitated through mineral-matrix interactions. Current data indicate an important role for non-collagenous extracellular matrix (ECM) 2 proteins associated with collagen fibrils in the process of biomineralization. The high affinity of these non-collagenous proteins for divalent cations may facilitate control of the nucleation and growth of the initial mineral deposits. In addition, these proteins may subsequently regulate the size, composition, microstructure, morphology, and the orientation of the resulting crystals formed (crystal growth) (1). Mineralized tissues are unique in their production of extracellular acidic phosphoproteins. These phosphoproteins may be of structural and/or regulatory significance to the formation of mineralized tissues, potentially acting as a key interface between the nucleating mineral crystal and surrounding collagen fibrils (2, 3). These phosphoproteins include: osteopontin (OPN) (4), bone sialoprotein (5, 6), dentin matrix protein 1 (DMP1) (7), phosphophoryn (PP) (8), osteonectin (9), bone acidic glycoprotein 75 kDa (BAG 75) (10), and MEPE (11), each accumulating in the extracellular matrices of bone and dentin to diffe...

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Characterization and Partial Purification of Microsomal Casein Kinase II from Osteoblast-like Cells: An enzyme that phosphorylates osteopontin and phosphophoryn

Cited by 6 publications

References 43 publications

Vascular smooth muscle cell calcification and SLC20 inorganic phosphate transporters: effects of PDGF, TNF-α, and Pi

Vascular smooth muscle cell calcification and SLC20 inorganic phosphate transporters: effects of PDGF, TNF-α, and Pi

Phosphorylation of the Proteins of the Extracellular Matrix of Mineralized Tissues By Casein Kinase-Like Activity

Expression of Phosphophoryn Is Sufficient for the Induction of Matrix Mineralization by Mammalian Cells

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