Protein kinase CK2 and new binding partners during spermatogenesis

Abstract

Protein kinase CK2 is an ubiquitously expressed enzyme that is absolutely necessary for the survival of cells. Besides the holoenzyme consisting of the regulatory β-subunit and the catalytic α- or α'-subunit, the subunits exist in separate forms. The subunits bind to a number of other cellular proteins. We show the expression of individual subunits as well as interaction with the transitional nuclear protein TNP1 and with the motor neuron protein KIF5C during spermatogenesis. TNP1 is a newly identified binding partner of the α-subunit of CK2. CK2α and KIF5C were found in late spermatogenesis, whereas CK2β and TNP1 were found in early spermatogenesis. CK2α, CK2α', TNP1, and KIF5C were detected in the acrosome of spermatozoa, while CK2β was detectable in the mid-piece. Combinations of CK2 subunits might determine interactions with other proteins during spermatogenesis. KIF5C as a kinesin motor neuron protein is probably involved in the redistribution of proteins during spermatogenesis.

Fig. 1

TNP1 binds to CK2α and the holoenzyme but not to CK2β and CK2α′. a Coomassie Blue-stained gel of the purified proteins used for the binding assay. b CK2α, CK2β, and CK2 holoenzyme consisting of CK2α₂β₂ were incubated with GST-tagged TNP1. After extensive washing, protein complexes were collected at GSH Sepharose. Bound proteins were analyzed on a 12.5% SDS polyacrylamide gel. CK2 subunits were identified with antibody #26 against CK2α and #32 against CK2β. c Protein A Sepharose-bound CK2α′-specific antibody #30 was incubated with GST-CK2α′ and then with GST-TNP1 (1) or GST-tag (2). As a control, protein A Sepharose-bound CK2α′-specific antibody #30 was incubated with GST-tag (3). Proteins were analyzed on a 12.5% SDS polyacrylamide gel and finally visualized with an antibody against the GST-tag. d Protein A Sepharose bound CK2α-specific antibody #30 was incubated with (1, 3) or without GST-CK2α′(2, 4) and then with GST-CK2β (3, 4) or with the GST-tag alone (1, 2). Proteins were analyzed on a 12.5% SDS polyacrylamide gel and finally visualized with an antibody against the GST-tag

Fig. 2

Influence of increasing concentrations of TNP1 on CK2 kinase activity. Purified CK2 holoenzyme was incubated with ³²PγATP with cyclin H as a substrate in the absence or presence of increasing concentrations of TNP1. As a control, we used the GST-tag or GST-TNP1. Phosphorylated proteins were analyzed on a 12.5% SDS polyacrylamide gel, followed by autoradiography

Fig. 3

Western blot analysis of CK2 subunits, TNP1, and KIF5C in spermatozoa. Spermatozoa extract (100 μg) was subjected to a 12.5% SDS polyacrylamide gel. After transfer of the proteins to a PVDF membrane, CK2α, CK2α′, CK2β, TNP1, and KIF5C were detected with the corresponding specific antibodies

Fig. 4

Immunolocalization of CK2 subunits, TNP1, and KIF5C in mouse testis. CK2α, CK2α′, CK2β, TNP1, and KIF5C were localized in mouse testis with DAB immunostaining. CK2α and KIF5C showed the same distribution during late spermatogenesis. The immunoreactions (visible in brown staining) appeared in the acrosome of spermatids. CK2α′ is also localized in late spermatogenesis. In addition to the acrosomal region, the staining is also visible in the cytoplasm. In contrast, CK2β was localized in early spermatogenesis in spermatogonia and spermatozytes. No immunoreactions were found with CK2β in later stages of spermatogenesis. TNP1 was also found in early spermatogenesis and in spermatogenetic cells, but was no longer displayed in elongated spermatids. Corresponding control stainings with an unspecific antibody are shown on the last picture of each row (bars 20 μm)

Fig. 5

Immunofluorescence analysis of CK2 subunits, TNP1, and KIF5C in spermatozoa. Epididymal spermatozoa were used for immunolocalization of CK2, TNP1, and KIF5C. CK2α, CK2α′, TNP1, and KIF5C antibodies labeled the acrosome of murine spermatozoa (Cy3/red). In addition, CK2α and CK2β immunoreactions were also detected in the mid-piece of the tail. On the right side of the panel, corresponding DIC images are shown. Control staining with an unspecific primary antibody did not show any immunoreactions (not shown; bar 20 μm)

Fig. 6

Transition electron microscopy of spermatids and spermatozoa for the detection of CK2 and KIF5C. Antibodies against CK2, TNP1, and KIF5C were used to identify the subcellular distribution of their corresponding proteins in spermatogenesis. Immunoreactions with the CK2α antibody are visible as gold particles (5 nm) in the developing acrosome of spermatids (a–c; c = magnification of b) and in the head of spermatozoa (d). KIF5C antibody labeled the developing acrosome of spermatids and spermatozoa (e–g) and mitochondria of the mid-piece (h). No, or only weak, immunoreactions were found with CK2α′, CK2β, and TNP1 antibodies with electron microscopy (not shown; bar 200 nm)