Temporal Association of Protamine 1 with the Inner Nuclear Membrane Protein Lamin B Receptor during Spermiogenesis

Abstract

During mammalian spermiogenesis, histones are replaced by transition proteins, which are in turn replaced by protamines P1 and P2. P1 protamine contains a short arginine/serine-rich (RS) domain that is highly phosphorylated before being deposited into sperm chromatin and almost completely dephosphorylated during sperm maturation. We now demonstrate that, in elongating spermatids, this phosphorylation is required for the temporal association of P1 protamine with lamin B receptor (LBR), an inner nuclear membrane protein that also possesses a stretch of RS dipeptides at its nucleoplasmic NH 2 -terminal domain. Previous studies have shown that the cellular protein p32 also binds tightly to the unmodified RS domain of LBR. Extending those findings, we now present evidence that p32 prevents phosphorylation of LBR and furthermore that dissociation of this protein precedes P1 protamine association. Our data suggest that docking of protamine 1 to the nuclear envelope is an important intermediate step in spermiogenesis and reveal a novel role for SR protein kinases and p32.The development of spermatids into spermatozoa, termed spermiogenesis, is characterized by the replacement of histones by the highly basic, arginine-rich, protamines (1). As a result of this exchange, the nucleosomal-type chromatin is transformed into a smooth fiber and compacted in a volume of about 5% of that of a somatic cell nucleus (2, 3). Although the exchange of chromatin proteins during spermiogenesis has long been known, the molecular mechanisms and the signaling pathways governing the histone to protamine transition have remained obscure.The deposition of protamines on sperm chromatin and the subsequent chromatin condensation appear to be controlled by phosphorylation-dephosphorylation events. Protamines are highly phosphorylated, shortly after their synthesis and before binding to DNA, whereas they become largely dephosphorylated during sperm maturation (4 -8). Phosphorylation of P2 protamine has been shown to be essential, because deletion of the calmodulin-dependent protein kinase Camk4, which phosphorylates P2 protamine, impairs the replacement of transition protein-2 with P2 protamine, resulting in defective spermiogenesis and male sterility (9). On the other hand, all P1 protamines contain short arginine/serine-rich (RS) 1 domains that are efficiently phosphorylated by SRPK1 (SR protein kinase 1) (10), but the physiological significance of this modification is mostly unknown.In this respect, Biggiogera et al. (11) reported that protamines initially appear at the nuclear periphery, implying that the nuclear envelope might play a role in the replacement of transition proteins by protamines during spermiogenesis. Given that RS domains mediate protein-protein interactions (12), we sought to investigate the potential interaction of P1 protamine with the inner nuclear membrane protein lamin B receptor (LBR), which also possesses a repeat of RS dipeptides at its nucleoplasmic NH 2 -terminal domain. In the present study we demonstrate a direct a...

Cytomegaloviral proteins that associate with the nuclear lamina: components of a postulated nuclear egress complex

“…Interestingly, pUL50 does not interact directly with the N-terminal cytoplasmic interaction platform of the LBR (aa 1-208; Gruenbaum et al, 2005). pUL53 and pUL97 were also negative for LBR interaction, which has been demonstrated previously (Mylonis et al, 2004;Marschall et al, 2005;Milbradt et al, 2007). The only protein that showed a positive reaction with LBR in this system was p32 (Fig.…”

“…In summary, the immunofluorescence experiments are consistent with the CoIP and yeast two-hybrid analyses, suggesting a complex formation among these viral and cellular proteins. In this context, p32 appears to be an important factor for establishing the NEC due to its various protein-protein interactions with pUL50, pUL53, pUL97, PKC and LBR (Milbradt et al, 2007;Marschall et al, 2005;Mylonis et al, 2004;Storz et al, 2000;Robles-Flores et al, 2002). Additionally, the recruitment of cellular and viral protein kinases to the nuclear rim is essential for the reorganization of the nuclear lamina (Muranyi et al, 2002;Park & Baines, 2006).…”

Non-random chromosome positioning in mammalian sperm nuclei, with migration of the sex chromosomes during late spermatogenesis

“…It might explain why they are released, but not their centralized location. Furthermore, protoamine 1, one of the proteins that complexes with DNA in sperm, has an affinity for LBR (Mylonis et al, 2004). Normally, in somatic cells, the chromatin-binding protein HP1 associates with LBR (Ye and Worman, 1996).…”

Lamin B receptor

“…More recently, evidence has been presented that phosphorylated protamine can bind to LBR during spermiogenesis in the rodent testis. 49 The authors view this observation as demonstrating that LBR plays a role in the orchestrated transition from histones to protamines, which may also involve the RS protein kinase 32 and p32. 30 A provocative recent study 50 presents evidence that a methylated DNA binding protein (MeCP2) binds directly to LBR, thus bringing certain heterochromatic regions into proximity to the NE.…”