A 180-kDa Protein Kinase Seems to Be Responsible for the Phosphorylation of Prothymosin α Observed in Proliferating Cells

Abstract

Prothymosin ␣ (ProT␣) is an acidic protein involved in cell proliferation. Its phosphorylation status is correlated with proliferative activity. Here we report the isolation and characterization of a ProT␣-phosphorylating kinase (ProT␣K) from mouse splenocytes that seems to be responsible for the in vivo phosphorylation of ProT␣ and that differs from other protein kinases reported to date. This enzyme, mainly located in the cytosol, has an molecular mass of 180 kDa and appears to be made up of two proteins of 64 and 60 kDa. Its activity was markedly enhanced by mitogenic activation of cells. The ProT␣ residues phosphorylated by the enzyme in vitro are a Thr at position 7 and another Thr at positions 12 or 13, both located within casein kinase 2 (CK-2) consensus motifs; these are the same residues as are phosphorylated in vivo. The new enzyme shows a number of clear structural and catalytic differences from CK-2. It phosphorylates histones H2B and H3, although with weaker activity than ProT␣. An enzyme with the same characteristics was also found in other murine tissues and cell lines.

Regulation of prothymosin α by estrogen receptor α: molecular mechanisms and relevance in estrogen-mediated breast cell growth

“…PTa has also been reported to be negatively regulated by p53, further supporting its potential role in proliferation of the cell (Zhao et al, 2000). The PTa protein is phosphorylated although the physiological relevance of this finding is not known (Perez-Estevez et al, 1997;Sburlati et al, 1993).…”

Prothymosin α Is Processed to Thymosin α1 and Thymosin α11 by a Lysosomal Asparaginyl Endopeptidase

“…The highly conserved primary structure of ProT␣, especially around the asparagine/glycine bonds cleaved by legumain (13), provides further support for the view that ProT␣ processing to yield T␣ 1 is generalized. Moreover, the present results indicate that processing of ProT␣ in vitro is independent of its phosphorylation state (ProT␣ is phosphorylated at specific residues when cells are activated to proliferate) (17,18). The presence of phosphorylated T␣ 1 in proliferating cells and the inability of the ProT␣ protein kinase to phosphorylate T␣ 1 (34) thus suggest that phospho-ProT␣ may be processed in vivo to yield phospho-T␣ 1 .…”

“…Dephosphorylated ProT␣ was obtained from this reaction mixture by RP-HPLC as indicated above. Phosphorylation of ProT␣ with radioactive orthophosphate was done using ProT␣ protein kinase, purified as described (18). Briefly, a phosphorylation reaction mixture containing 50 g of dephosphorylated ProT␣, 50 mM Tris-HCl, pH 7.4, 150 mM KCl, 26 mM MgCl 2 , 1.6 mM EGTA, 3.3 mM DTT, 80 ng/ml protamine, 83 mM ␤-glycerol phosphate, and 100 mM [ 32 P]ATP (3,000 Ci/mmol) in a total volume of 250 l was incubated at 37°C for 45 min.…”

“…Its wide distribution in mammalian tissues suggested that its function was probably not immunological, despite the apparent immunoregulatory properties of T␣ 1 and T␣ 11 . Subsequent studies indicated that ProT␣ has a generalized role in the proliferation of mammalian cells, by mechanisms involving migration to the nucleus (14 -16) and cytosolic phosphorylation of ProT␣ (17,18). Reports from our group and others over recent years have provided increasing evidence that the nuclear function of ProT␣ involves interactions with histones (19 -21) and with other proteins related to DNA metabolism, including proliferating cell nuclear antigen, Cdk2, and cyclin A (22), arguing strongly for a role in chromatin remodeling.…”

Multiple tRNA attachment sites in prothymosin α

“…A choice of ProTα modification via RNA linking, in addition to phosphorylation which has also been described for ProTα [26–28], is notable. A likely explanation for this phenomenon would be the participation of ProTα in some aspect of tRNA metabolism.…”