[Frontiers in Bioscience 2, a26-30, September 15, 1997]
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CAVEAT LECTOR




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SPERMINE STIMULATES THE PHOSPHORYLATION OF THE NUCLEAR MATRIX PROTEINS CATALYZED BY NUCLEAR KINASE II

Rati Verma and Kuang Yu Chen

Department of Chemistry and The Cancer Institute of New Jersey, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08855-0939

Received 8/19/97 Accepted 8/25/97

4. RESULTS

4.1 Purification and characterization of the NII nuclear kinase

Nuclear protein kinase NII from NB-15 cells was purified 706-fold over the total casein kinase activity present in the nucleoplasm after a consecutive phosphocellulose column chromotography. The specific activity of the purified NII kinase was 100 units per microgram protein (1 unit=amount of enzyme catalyzing the transfer of 1 pmol of phosphate from ATP to dephosphorylated casein per min at 300 C, pH 7.4). When the NII kinase isolated from NB-15 cells was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining, three bands of apparent molecular weights, 44,000, 40,000, and 26,000 were observed (data not shown). This subunit composition matches that reported for casein kinase II purified from other sources (16). When NII kinase activity was measured at saturating concentrations of casein (2 mg/ml), a Km value of 13 micromolar was found for ATP. Similarly, a Km value of 0.85 mg/ml and Vmax value of 5.3 pmol/min was found for casein when NII kinase activities were measured at saturating concentration of ATP (50 micromolar). These values are in agreement with that reported for casein kinase II isolated from other sources (1, 16). Among the three commonly used non-physiological substrates for kinase assay, NII kinase phosphorylated both alpha-casein and phosvitin but not histone (data not shown). Spermine at 1- 2 mM only stimulated the phosphorylation of alpha-casein but not phosvitin. Kinetic measurement indicated that upon addition of 2 mM spermine, the Km value of NII kinase for alpha-casein was reduced by 11% to 0.76 mg/ml but Vmax was almost doubled to 10.4 pmols/min (data not shown). Among the polyanions examined, heparin was the most potent inhibitor which completely abolished NII activity at a concentration of 0.5-1 mg/ml (Table 1). The inhibition could be reversed by spermine at 2 mM (Table 1). Poly (A), poly (U), and calf thymus DNA also inhibited NII activity but were less effective than heparin (Table 1). Interestingly the addition of spermine to the assay mixture reversed the inhibitory effects of poly (A) and cal thymus DNA, but not that of poly (U), indicating that factors other than counterion interaction were involved in the action of spermine.

TABLE 1. Effects of various polyanions on the activity of nuclear NII protein
kinase isolated from NB-15 mouse neuroblastoma cells

POLYANION (microgram/ml)

SPERMINE (1 mM)

% ACTIVITY

Heparin (0.1)

-

35

Heparin (0.5)

-

19

Heparin (0.5)

+

111

Poly (U) (150)

-

7

Poly (U) (150)

+

17

Poly (A) (150)

-

80

Poly (A) (150)

+

143

DNA (150)

-

46

DNA (150)

+

135

The chemicals to be tested were added to the standard phosphorylation assay mixture for NII kinase as described in "Experimental Procedures". Results are the average of dulplicate measurements, expressed as per cent of control in the absence of added polyanions.

4.2 NII kinase catalyzed phosphorylation of nuclear proteins

Although casein and phosvitin have been commonly used as protein substrates for NII kinase (or casein kinase II), neither of which are nuclear proteins. To search for possible physiological protein substrates, we first examined the phosphorylation pattern by adding NII kinase to nuclear extracts. Figure 1 (lane B vs lane A) shows that addition of NII did not significantly alter the endogenous protein phosphorylation pattern due to high endogenous protein kinase activity in nucleoplasms. However, brief heat treatment completely abolished all endogenous kinase activities (Figure 1, lane C), but did not affect the ability of nuclear proteins to function as substrates for exogenously added NII kinase (Figure 1, lane D). The nuclear protein phosphorylation pattern generated by NII kinase closely resembled that of the endogenous phosphorylation except in the low molecular weight region (Figure 1, lanes D vs A). Spermine at 1mM significantly stimulated the NII kinase catalyzed phosphorylation of nuclear proteins (Figure 1, lanes E vs D).

Figure1. Nuclear protein phosphorylation by endogenous and exogenously added nuclear kinases. Nucleoplasm without (lanes A and B) or with (lanes C-E) prior heat treatment was added to the phosphorylation assay mixture. The final protein concentration was 4 mg per ml. Lane A, no addition; lane B, 6 units NII added; lane C, no addition; lane D, 8 units of NII kinase added; lane E, 8 units of NII kinase and 1 mM sperimne added.

4.3 Phosphorylation of subnuclear fractions by NII nuclear kinase

To further examine the nature of these phosphorylated nuclear proteins, nuclei were fractionated into three subnuclear fractions: (i) the high mobility group (HMG), (ii) the heterogeneous nuclear ribonucleoprotein (hnRNP) and (iii) the nuclear matrix proteins. HMG represents a family of architectural proteins that may be involved in transcriptional regulation of genes (17). The effects of spermine and heparin on NII catalyzed phosphorylation of HMG proteins are shown in Figure 2. The addition of NII kinase to the HMG fraction resulted in the phosphorylation of a number of HMG proteins (Figure 2, lane A). The most prominently phosphorylated protein substrates of NII kinase were of apparent Mr 46,000, 20,000, 18,000, and 16,000 (Figure 2, lane A). The 20,000, 18,000, and 16,000-dalton bands were identified as HMG 14a, 14b, and 17 protein respectively based on their position on the 15% sodium dodecyl sulfate-polyacrylamide gel (18). Addition of spermine up to 6 mM had neither stimulatory nor inhibitory effect on the phosphorylation of HMG proteins catalyzed by NII kinase (Figure 2, lanes B to E). Addition of heparin (0.5 mg/ml) also had no effect on the NII catalyzed phosphorylation of HMG proteins (Figure 2, lane G). Interestingly, HMG proteins are very poor substrates for NI kinase (14).

Figure2. Effect of spermine on phosphorylation of the HMG proteins by NII kinase. The HMG fraction (16 microgram proteins) was added to the standard assay mixture containing 30 g/ml bovine serum albumin and 6 units of NII kinase. Activity was assayed in the presence of the following additions: Lane A, no addition; lane B. 1 mM spermine; lane C, 2 mM spermine; lane D, 4 mM spermine; lane E, 6 mM spermine; lane F, 10 mM putrescine; and lane G, 0.5 microgram/ml heparin. In lane H, HMG fraction alone was added to the assay mixture, no NII kinase added.

Heterogeneous nuclear ribonucleoproteins or hnRNPs represent a class of about 20 major polypeptides that may be involved in post-transcriptional events such as regulated splicing and mRNA export (19). The hnRNP fraction contained high endogenous kinase activity (Figure 3, lane A). Brief heat treatment completely abolished the endogenous kinase activities (Figure 3, lane B). The addition of NII kinase added to the heated hnRNP fraction phosphorylated many proteins and yield an almost identical phosphorylation pattern as that of endogenous phosphorylation (Figure 3, lanes C vs A). Spermine had a slight stimulatory effect and heparin had a slight inhibitory effect on the action of NII kinase on hnRNP phosphorylation (Figure 3, lanes D and E vs C).

Figure 3. Effects of spermine andheparinon the phosphorylationofthehnRNP fraction byNIIKinase. Lane A, endogenous phosphorylation of hnRNP. Heat-treated hnRNP fraction (2 mg/ml) was used in lanes B-E: lane B, no addition; lane C, 6 units NII kinase added; lane D, 6 units NII kinase and 2 mM spermine added; lane E, 6 units NII kinase and 10 microgram/ml heparin added.

The nuclear matrix fraction isolated from NB-15 cells included proteins of apparent Mr of 110,000 72,000, 68,000 and 62,000 and exhibited similar protein profile as that prepared from other sources (20). Nuclear matrix represents a structural framework of the nucleus and is implicated in DNA replication and RNA processing (21). The endogenous kinase activity associated with the nuclear matrix fraction was insignificant (Figure 4, lane A). Addition of NII kinase to the unheated nuclear matrix resulted in the phosphorylation of many proteins, most notably, a 55,000-dalton protein (Figure 4, lane B). Spermine at 2 mM stimulated the NII kinase-catalyzed the phosphorylation of all these proteins (Figure 4, lane C). The stimulatory effect of spermine was significant and estimated to be more than 5-fold based on densitometric tracing. Addition of 10 mg/ml heparin resulted in significant inhibition of the phosphorylation (Figure 4, lane D).

Figure 4. Effects of spermine and heparin on the phosphorylation of nuclear matrix proteins byNIIkinase. Nuclear matrix (0.7 mg/ml protein) was added to the standard assay mixture containing 30 microliter/ml bovine serum albumin. Lane A, nuclear matrix fraction alone; lane B, 4 units NII kinase added; lane C, 4 units NII kinase and 2 mM spermine added; lane D, 4 units NII kinase and 10 microliter/ml heparin added.