Intermediate filament reconstitution in vitro. The role of phosphorylation on the assembly-disassembly of desmin.

Self Cite

Abstract. We Z 11:2895-2902). This observation implies a possibility that there is a protein kinase specifically activated at metaphaseanaphase transition. To further analyze the cell cycledependent GFAP phosphorylation, we prepared monoclonal antibodies KT13 and KT34 which recognize the phosphorylation of GFAP at Serl3 and Ser34, respectively. Immunocytochemical studies with KT13 and KT34 revealed that the GFAP phosphorylation in the cleavage furrow during late mitotic phase occurred not only in glioma cells but also in human SW-13 and mouse Ltk-cells in which GFAP was ectopically expressed, thus the phosphorylation can be monitored in a wide range of cell types. Furthermore, we detected kinase activity which phosphorylates GFAP at Serl3 and Ser34 in the lysates of late mitotic cells but not in those of interphase cells or early mitotic cells. These results suggest that there exists a protein kinase which is specifically activated at the transition of metaphase to anaphase not only in GFAP-expressing cells but also in cells without GFAP.

Section: Discussionmentioning

confidence: 99%

Detection of protein kinase activity specifically activated at metaphase-anaphase transition.

Sekimata

Tsujimura

Tanaka

et al. 1996

Self Cite

“…Numbers of observations strongly suggest that phosphorylation of vimentin may regulate its morphological organization. In vitro studies reported that site-specific phosphorylation of vimentin induced disassembly of filament structure (Inagaki et al, 1987(Inagaki et al, , 1988Evans, 1988). In vivo experiments demonstrated that increase in vimentin phosphorylation by microinjection of the catalytic subunit of A kinase (Lamb et al, 1989), mitotic arrest with colcemid treatment (Franke et al, 1982;Chou et al, 1990;Tsujimura et al, 1994), or treatment with phosphatase inhibitors (Eriksson et al, 1992) led to the collapse of vimentin filaments into bundles and granular dot structures.…”

Section: Discussionmentioning

confidence: 99%

Differential targeting of protein kinase C and CaM kinase II signalings to vimentin.

Ogawara

Inagaki

Tsujimura

et al. 1995

Abstract. Hydrolysis of inositol phospholipids by receptor stimulation activates two separate signaling pathways, one leading to the activation of protein kinase C (C kinase) via formation of diacylglycerol. The other is the inositol trisphosphate (IP3)/Ca 2÷ pathway and a major downstream kinase which is activated is Ca2÷/calmodulin-dependent protein kinase II (CaM kinase II). To examine signaling pathways of C kinase and CaM kinase II to the cytoskeletal protein vimentin, we prepared monoclonal antibodies YT33 and MO82 which recognize the phosphorylation state of vimentin by C kinase and by CaM kinase II, respectively. Ectopic expression of constitutively active C kinase or CaM kinase II in primary cultured astrocytes by microinjection of the corresponding expression vectors induced phosphorylation of vimentin at each specific phosphorylation site, followed by reorganization of vimentin filament networks. In contrast, simultaneous activation of C kinase and CaM kinase II by inositol phospholipid hydrolysis with receptor stimulation led to an exclusive phosphorylation of vimentin at the CaM kinase II site, not at the site of C kinase. These results indicate that the intracellular targeting of C kinase and CaM kinase II signalings to vimentin is regulated separately, under physiological conditions. "r~ECEPTOR-mediated hydrolysis of phosphatidylinosi-1 ~" tol 4,5-bisphosphate (PIP2) relays extracellular l ~. signals into the cell and two kinases, protein kinase C (C kinase) and Ca2÷/calmodulin-dependent protein kinase II (CaM kinase II) play a central role in this signaling. Hydrolysis of PIP2 leads to formation of diacylglycerol and activation of inositol trisphosphate (IP3)-induced Ca 2+ signaling (for reviews see Michell, 1992;Berridge, 1993). C kinase and CaM kinase II, activated by diacylglycerol and Ca 2+, respectively, transmit these two signalings to downstream molecules (for reviews see Nishizuka, 1984Nishizuka, , 1986Colbran et al., 1989; Hanson and Schtflman, 1992). From the viewpoint of signaling cascade, one of the most distinctive features of kinases represented by C kinase and CaM ki-

“…Its physical association with the IF CK8 and 18 and their phosphorylation in situ points to such a possibility. Although the role of phosphorylation in intermediate filament function is not fully understood, phosphorylation does appear to play a role in the assembly and disassembly of vimentin and desmin (Inagaki et al ., 1987(Inagaki et al ., , 1988 . Alternatively, binding of the fragment to the cytokeratin may protect the catalytic domain from further degradation and provide a ki- Immunofluorescent staining of HT29 cells using both anti-CK8/18 and anti-PKCe antibodies showed a similar staining profile with immunofluorescence seen in both the cell surface and cytoplasm .…”

Section: Discussionmentioning

confidence: 99%

PKC epsilon-related kinase associates with and phosphorylates cytokeratin 8 and 18.

Omary

Baxter

Chou

et al. 1992

Abstract. A 40-kD protein kinase C (PKC)E related activity was found to associate with human epithelial specific cytokeratin (CK) polypeptides 8 and 18. The kinase activity coimmunoprecipitated with CK8 and 18 and phosphorylated immunoprecipitates of the CK. Immunoblot analysis of CK8/18 immunoprecipitates using an anti-PKCE specific antibody showed that the 40-kD species, and not native PKCE (90 kD) associated with the cytokeratins. Reconstitution experiments demonstrated that purified CK8 or CK18 associated with a 40-kD tryptic fragment of purified PKCE, or with a similar species obtained from cells that express the fragment constitutively but do not express CK8/18. C YTOKERATINS (CK)' are a group of intermediate filament (IF) proteins which are expressed primarily in epithelial tissues (Lazarides, 1982 ;Steinert and Roop, 1988 ;Franke et al ., 1981;Osborn and Weber, 1986) . The 30 or so polypeptides which make up the family ofcytokeratin proteins are divided into acidic (type I) and basic/ neutral (type II) keratins. In epithelial cells, CK are found as mosaic noncovalent polymers with an assembly consisting of at least one type I and one type II CK (Steinert and Roop, 1988). For example, "simple" single layer epithelial cells such as intestinal epithelia express CK8 (type II) and CK18 (type I), whereas esophageal epithelial cells express CK4 (type II) and CK13 (type I) predominantly. Cytokeratins are not only important as tissue-specific markers, they also form important markers of cell differentiation .IF proteins, including cytokeratins, undergo several posttranslational modifications such as N112-terminal acetylation (Steinert and Idler, 1975), glycosylation (King and Hounsell, 1989;Roberts and Brunt, 1986), and serine/threonine phosphorylation (Steinert, 1988;Gilmartin et al ., 1984 ;Yeagle et al., 1990;Baribault et al., 1989) . The functional role of IF protein phosphorylation is not well under- A peptide pseudosubstrate specific for PKCE inhibited phosphorylation of CK8/18 in intact cells or in a kinase assay with CK8/18 immunoprecipitates . Tryptic peptide map analysis -of the cytokeratins that were phosphorylated by purified rat brain PKCE or as immunoprecipitates by the associated kinase showed similar phosphopeptides . Furthermore, PKCE immunoreactive species and CK8/18 colocalized using immunofluorescent double staining . We propose that a kinase related to the catalytic fragment of PKCE physically associates with and phosphorylates cytokeratins 8 and 18.