Casein kinase II motif-dependent phosphorylation of human papillomavirus E7 protein promotes p130 degradation and S-phase induction in differentiated human keratinocytes

The E7 proteins of human papillomaviruses (HPVs) promote S-phase reentry in differentiated keratinocytes of the squamous epithelia to support viral DNA amplification. In this study, we showed that nuclear p130 was present in the differentiated strata of several native squamous epithelia susceptible to HPV infection. In contrast, p130 was below the level of detection in HPV-infected patient specimens. In submerged and organotypic cultures of primary human keratinocytes, the E7 proteins of the high-risk mucosotrophic HPV-18, the benign cutaneous HPV-1, and, to a lesser extent, the low-risk mucosotropic HPV-11 destabilized p130. This E7 activity depends on an intact pocket protein binding domain and a casein kinase II (CKII) phosphorylation motif. Coimmunoprecipitation experiments showed that both E7 domains were important for binding to p130 in extracts of organotypic cultures. Metabolic labeling in vivo demonstrated that E7 proteins were indeed phosphorylated in a CKII motif-dependent manner. Moreover, the efficiencies of the E7 proteins of various HPV types or mutations to induce S-phase reentry in spinous cells correlated with their relative abilities to bind and to destabilize p130. Collectively, these data support the notion that p130 controls the homeostasis of the differentiated keratinocytes and is therefore targeted by E7 for degradation to establish conditions permissive for viral DNA amplification.     

The E7 protein of human papillomavirus type 16 is phosphorylated by casein kinase II

The E7 protein of human papillomavirus type 16 (HPV16) transforms cultured cells and cooperates with the ras or fos oncogenes in the transformation of primary cells. In this study we have investigated the phosphorylation of E7. When we immunoprecipitated E7 from CaSki cells with a rabbit polyclonal antiserum to a bacterial fusion protein (trpE-E7), we found that E7 was phosphorylated at serine residues contained in five characteristic thermolysin peptides. Immunoprecipitated E7, and fusion proteins harboring the E7 protein from various HPV types, could all be specifically phosphorylated in vitro by the ubiquitous, growth factor-activated casein kinase II (CKII). Comparative peptide mapping showed that the sites of in vivo and in vitro phosphorylation are the same. CKII was shown previously to specifically phosphorylate serine or threonine residues within a cluster of acidic amino acids. The E7 protein contains such a sequence between amino acids 30 and 37. When a synthetic peptide corresponding to this region of E7 was phosphorylated by CKII in vitro, its thermolysin digestion products were the same as those in the phosphorylated E7 protein. We conclude that E7 is phosphorylated in vivo only at serines within the predicted CKII site and that CKII, or a CKII-like enzyme, participates in the reaction. Both the E1A and SV40 large T proteins contain similar CKII consensus sites proximal to the regions required for their associations with the retinoblastoma gene product (p105Rb). Thus it is conceivable that CKII phosphorylation can modulate the interaction between the transforming proteins and the retinoblastoma gene product.     

Casein kinase II phosphorylation of caldesmon downregulates myosin-caldesmon interactions

It is well-known that caldesmon (CaD) is a substrate for casein kinase II (CKII), and the phosphorylation of CaD by CKII regulates the interaction of CaD with myosin. However, the functionally relevant CKII phosphorylation site(s) on CaD and the precise role of CaD phosphorylation by CKII in mediating CaD's function have remained elusive. In this study, we demonstrate that Ser-26 is the major CKII phosphorylation site on CaD, while Ser-73 is of relatively minor importance. Moreover, the phosphorylation of Ser-26 and Ser-73 reduced CaD's ability to bind myosin by 45% and 27%, respectively, suggesting that the interaction of CaD with myosin is downregulated, at least in part, by the phosphorylation of these serine residues by CKII. Our results also demonstrate that there are at least four myosin-binding motifs within the amino-terminal region of CaD, located between residues 1-23, 34-43, 44-53, and 86-115, respectively. The myosin-binding motif between residues 44-53 contributes to strong myosin binding, while the three other myosin-binding motifs are responsible for weak myosin binding. The sequences between residues 24-33 and 54-85 on CaD are not required for the binding of CaD to myosin; thus, both Ser-26 and Ser-73 are located outside of the myosin-binding motifs. It is therefore likely that the downregulation of myosin-CaD interactions by CKII phosphorylation is due to phosphorylation-induced conformational changes in the adjacent myosin-binding motifs on CaD, rather than by the direct modification of these myosin-binding motifs by CKII.     

Casein kinase II activity is required for transferrin receptor endocytosis.

The effect of protein kinase inhibitors on transferrin receptor (TR) internalization was examined in HeLa, A431, 3T3-L1 cells, and primary chicken embryo fibroblasts. We show that TR endocytosis is not affected by tyrosine kinase or protein kinase C inhibitors, but is inhibited by one serine/threonine kinase inhibitor, H-89. Inhibition occurred within 15 min, was completely reversible after H-89 withdrawal, and was specific for endocytosis rather than pinocytosis since a TR mutant lacking an internalization signal was not affected. Interestingly, H-89 also inhibited the internalization of a TR chimera containing the major histocompatibility complex class II invariant chain cytoplasmic tail, indicating that the effect was not specific for the TR. Since H-89 inhibits a number of kinases, we employed a permeabilized cell endocytosis assay to further characterize the kinase. In permeabilized 3T3-L1 cells, addition of pseudosubstrate inhibitor peptides of casein kinase II (CKII) blocked TR internalization by more than 50%, whereas pseudosubstrates of cyclic AMP-dependent kinase A, protein kinase C, and casein kinase I had no effect. Furthermore, addition of purified CKII to the cell-free reactions containing CKII pseudosubstrates reversed the endocytosis block, suggesting that CKII or a CKII-like activity is required for constitutive endocytosis.     

Casein kinase II and the tumor suppressor protein P53 associate in a molecular complex that is negatively regulated upon P53 phosphorylation.

Selective immunoisolation of P53 from Sf9 cells coexpressing wild-type P53 and casein kinase II yielded a preparation containing casein kinase II, thus suggesting that the two proteins may associate in a molecular complex in the intact cell. Such a complex could indeed be demonstrated in vitro between purified recombinant P53 and oligomeric casein kinase II and was shown to dissociate when P53 became phosphorylated by the kinase. This suggested that the P53 C-terminal domain, which contains the casein kinase II phosphorylation site was involved in the protein-protein interaction; this was confirmed by the fact that an anti-P53 monoclonal antibody directed to that domain inhibited the P53-casein kinase II association. Studies with isolated recombinant casein kinase II subunits disclosed that although the alpha (catalytic) subunit could phosphorylate P53, the formation of a stable P53-casein kinase II association required the presence of the beta subunit of the kinase. This was confirmed by immunoisolation of a P53-beta subunit complex from cells expressing both polypeptides. Although the biological significance of a reversible P53-casein kinase II molecular complex in the control of cell proliferation processes remains to be defined, these observations suggest the possibility of a novel mechanism regulating P53 and casein kinase II activities in the intact cell.     

Casein kinase II phosphorylation of signal sequence receptor alpha and the associated membrane chaperone calnexin.

Signal sequence receptor alpha (SSR alpha) and calnexin are major calcium-binding proteins of the endoplasmic reticulum (ER) which are implicated in chaperone functions. They were identified as major membrane substrates after in vitro phosphorylation of ER membranes with [gamma-32P]GTP (Wada, I., Rindress, D., Cameron, P. H., Ou, W. J., Doherty, J.-J., II, Louvard, D., Bell, A. W., Dignard, D., Thomas, D. Y., and Bergeron, J. J. M. (1991) J. Biol. Chem. 266, 19599-19610). Using purified SSR alpha and associated calnexin as substrates, we have attempted to identify the kinase(s) responsible for their phosphorylation. A salt extract from canine pancreatic ER membranes and cytosol possessed SSR alpha kinase activity which showed identical chromatographic behavior through phosphocellulose, DEAE-Sepharose, and hydroxylapatite purification protocols. Final purification was effected from the cytosol with three polypeptides of 38, 36, and 28 kDa detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On the basis of primary sequence analysis of the three subunits of the purified kinase and the reconstitution of phosphorylation of SSR alpha and associated calnexin in heat-inactivated ER membranes by the addition of the purified kinase we conclude that the ER-associated kinase responsible for the GTP phosphorylation of SSR alpha and associated calnexin is casein kinase II.     

Casein kinase II activity and polyamine-stimulated protein phosphorylation of cytosolic and plasma membrane proteins in bovine sperm

A highly purified preparation of sperm cytosolic protein kinase was obtained by repeated chromatography with phosphocellulose. The preferred substrate of the enzyme was casein and the activity was not stimulated by added Ca2+, calmodulin, or cAMP. With casein as substrate, both ATP and GTP served as phosphate donors and the activity was inhibited by low micromolar heparin and stimulated by low millimolar spermine and spermidine. These properties are characteristic of casein kinase II from other cells. Endogenous protein substrates of the enzyme in sperm cytosolic fractions and in plasma membranes were demonstrated by incubating the preparations with [gamma-32P]GTP, under conditions unfavorable to other protein kinases, and analyzing the products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Spermine greatly enhanced the phosphorylation of three (55, 92, and 106 kDa) proteins in both cytosolic and plasma membrane preparations. Our results indicate that polyamines play a role in modulating the phosphorylation state of proteins in sperm and may further regulate sperm function through this mechanism.     

Casein kinase II accumulation in the nucleolus and its role in nucleolar phosphorylation

A rabbit antiserum against highly purified casein kinase II from mouse tumor cells was used for immunolocalization of the enzyme in fixed, permeabilized mouse cells. Casein kinase II was highly accumulated in nucleoli compared to the extra-nucleolar space of the nucleus or to the cytoplasma. Casein kinase II samples highly purified from the cytoplasma, from the extra-nucleolar fraction of the nucleus or from nucleoli exhibited no differences with respect to structure and function. All samples originally had an alpha 2 beta 2 structure (alpha, 42 kDa; beta, 24 kDa) showing formation of the alpha'-chain (36 kDa) only in the late steps of purification. The isoelectric point of the alpha-chain of all three samples was pH 7.7 and that of the beta-chain was pH 6.4-6.6. Using ATP or GTP, all three casein kinase II samples gave the same results of maximum phosphorylation of purified nucleolar marker phosphoproteins pp105/C23, pp135 and B23, yielding pp135 as one of the most highly phosphorylated proteins with an incorporation of about 75 phosphate groups per molecule pp135. Studies on optimum conditions of phosphorylation of nucleolar phosphoproteins by casein kinase II revealed that each of the protein substrates individually responded to alterations of assay parameters such as pH, magnesium ion and sodium chloride concentrations indicating that predominantly individual structural criteria were responsible for optimum phosphorylation. The determination of the apparent Km of casein kinase II for purified nucleolar phosphoproteins yielded values of 0.15 microM (pp105/C23), 0.1 microM (pp135) and 1.0 microM (B23) identifying them as high-affinity substrates of casein kinase II.     

Casein kinase II is a major protein phosphorylating activity in the nuclei of Xenopus laevis oocytes

The nuclei of Xenopus laevis oocytes contain kinases capable of phosphorylating endogenous and exogenous proteins using either ATP or GTP as phosphoryl donors. These enzymes are much more active with casein and phosvitin as substrates than with histones or protamines. The protein phosphorylating activity of oocyte nuclear extracts is not regulated by cyclic nucleotides, phorbol esters, calmodulin and calcium, or phospholipids. However, the casein phosphorylating activity can be greatly enhanced by the polyamines spermine or spermidine and drastically inhibited by heparin. Fractionation of the nuclear casein kinase activities by DEAE-Sephadex chromatography and glycerol gradient centrifugation indicate that the nuclei contain enzymes with the properties of casein kinases I and II as characterized in other species. Oocyte casein kinase I (Mr 37,000) is specific for ATP as phosphoryl donor, is only slightly inhibited by 10 micrograms/ml heparin, and is not significantly stimulated by polyamines. Casein kinase II (Mr 135,000) can use both ATP and GTP as substrates, and is very sensitive to heparin inhibition and polyamine stimulation. The fact that low concentrations of heparin (10 micrograms/ml) can inhibit a large percentage of the endogenous phosphorylation of nuclear extracts or of whole nuclei indicates that casein kinase II is probably the major protein phosphorylating activity of these oocyte organelles.     

Casein kinase II is elevated in solid human tumours and rapidly proliferating non-neoplastic tissue

Protein kinase CKII (i.e. casein kinase II, CKII, NII) is expressed at a higher level in rapidly proliferating tissues and in solid human tumours (e.g. colorectal carcinomas) when compared to the corresponding non-neoplastic colorectal mucosa. This could be shown by (a) Western blotting of cellular extracts from solid tumours followed by immunostaining with an anti-CKII polyclonal antibody, (b) immunohistochemical staining of cells from tissue sections and (c) by activity measurements using the CKII-specific synthetic peptide (RRRDDDSDDD). The maximum observed activity in the colorectal carcinomas investigated was up to eightfold higher in the tumour specimens than in the non-neoplastic tissue (i.e. colorectal mucosa). The activity range was between 33-350 U/mg protein and in the case of colorectal mucosa 13-106 U/mg protein. The amount of CKII determined in the individual tumours was in the range 0.4-1.6 nmol/g tissue.     

Identification of B-epitopes in the human papillomavirus 18 E7 open reading frame protein.

A panel of murine mAb raised against a MS2 replicase/HPV 18 E7 fusion protein included 23 reactive by ELISA with HPV 18 E7 determinants. A total of 19 of the 23 recognized linear epitopes in the N-terminal region of the E7 molecule, while the other four were deduced by binding inhibition assays to recognize conformational determinants in this region. All tested antibodies precipitated a 14-kDa peptide doublet that corresponded with the predicted size of the E7 protein, from HeLa cells, but not from HPV 16 E7 containing CaSki cells. HPV 18 E7 protein was detected by immunolabeling with electron microscopy in both the nucleus and the cytoplasm of HeLa cells with the greater proportion occurring in the cytoplasm. No antibody reacted specifically by indirect immunofluorescence with HeLa cells. Weak cross-reactivity of some mAb with the E6 MS2-replicase fusion protein of HPV 16 was detected by ELISA, but no protein of the appropriate size was immunoprecipitated from CaSki cells. It is concluded that the B cell epitopes on the HPV 18 E7 transforming protein are located in the N-terminal region of the molecule and that some are weakly cross-reactive with HPV 16 E6 protein. E7 protein is either present in HeLa cells at a concentration too low to be detected by indirect immunofluorescence, or the N-terminal epitopes are masked by protein conformation or interaction with cellular or other viral components.     

Tyrosine protein phosphorylation is required for protein kinase C-mediated proliferation in T cells.

We have studied the role of tyrosine kinase in PMA-stimulated T cells. Protein kinase C (PKC)-mediated D10A cell proliferation is inhibited by the specific inhibitor of tyrosine kinase, tyrphostin. This inhibitor selectively blocks the mRNA expression of the proto-oncogene c-myc in response to the phorbol ester, PMA. On the other hand, the same doses of this inhibitor do not affect the mRNA expression of the proto-oncogene c-fos in PMA-stimulated D10A cells. Phorbol esters induce in this T cell line the tyrosine phosphorylation of a unique protein of 42 kDa and the enzyme PKC is required for this activity.     

Arginine-glycine-aspartic acid motif is critical for human parechovirus 1 entry

The human parechovirus 1 RGD motif in VP1 was studied by mutagenesis. An RGD-to-RGE change gave only revertant viruses with a restored RGD, while deletion of GD was lethal and nonrevertable. Mutations at the +1 and +2 positions had some effect on growth properties and a +1 M-to-P change was lethal. These studies indicate that the RGD motif plays a critical role in infectivity, presumably by interacting with integrins, and that downstream amino acids can have an influence on function.     

NEK7 is a centrosomal kinase critical for microtubule nucleation

NIMA in Aspergillus nidulans is a mitotic kinase for chromosome condensation and segregation. We characterized NEK7, a human homologue of Aspergillus NIMA. NEK7 was located at the centrosome throughout the cell cycle. Temporal localization of NEK7 at midbody of the cytokinetic cell was also observed. NEK7 knockdown by RNAi caused a prometaphase arrest of the cell cycle with monopolar or disorganized spindle. We propose that such defects in spindle assembly are resulted from reduction in microtubule nucleation activity at the centrosome. In consistent to the proposal, we observed a decrease in the centrosomal gamma-tubulin levels and reduction of the microtubule re-growth activity in the NEK7-suppressed cells. In addition, it was evident that NEK7 was directly involved in cytokinesis.