Mitotic Regulation of Protein 4.1R Involves Phosphorylation by cdc2 Kinase

Abstract

The nonerythrocyte isoform of the cytoskeletal protein 4.1R (4.1R) is associated with morphologically dynamic structures during cell division and has been implicated in mitotic spindle function. In this study, we define important 4.1R isoforms expressed in interphase and mitotic cells by RT-PCR and mini-cDNA library construction. Moreover, we show that 4.1R is phosphorylated by p34cdc2 kinase on residues Thr60 and Ser679 in a mitosis-specific manner. Phosphorylated 4.1R135 isoform(s) associate with tubulin and Nuclear Mitotic Apparatus protein (NuMA) in intact HeLa cells in vivo as well as with the microtubule-associated proteins in mitotic asters assembled in vitro. Recombinant 4.1R135 is readily phosphorylated in mitotic extracts and reconstitutes mitotic aster assemblies in 4.1R-immunodepleted extracts in vitro. Furthermore, phosphorylation of these residues appears to be essential for the targeting of 4.1R to the spindle poles and for mitotic microtubule aster assembly in vitro. Phosphorylation of 4.1R also enhances its association with NuMA and tubulin. Finally, we used siRNA inhibition to deplete 4.1R from HeLa cells and provide the first direct genetic evidence that 4.1R is required to efficiently focus mitotic spindle poles. Thus, we suggest that 4.1R is a member of the suite of direct cdc2 substrates that are required for the establishment of a bipolar spindle.

Tastin is required for bipolar spindle assembly and centrosome integrity during mitosis

“…Our current data also demonstrated that tastin is phosphorylated in mitosis, similar to Protein4.1 and NuMA (15,34). A motif analysis in the ELM database shows that tastin contains many potential sites for serine/threonine phosphorylation by protein kinases, including CDK1, polo-like kinase, and mitogen-activated protein kinase, which are highly activated during mitosis (35).…”

“…Transient transfection was carried out with calcium phosphate, FuGENE 6 Transfection Reagent (Roche Diagnostics, Mannheim, Germany), or electroporation (Eppendorf, Germany). HeLa cell synchronization was achieved as described previously (15). Briefly, synchronization at the G1/S boundary was achieved by the thymidine (2.5 mM, Sigma, St. Louis, MO, USA) double block technique; an S‐phase sample was collected 3.5 h after release from the second thymidine block.…”

Alternatively spliced exon 5 of the FERM domain of protein 4.1R encodes a novel binding site for erythrocyte p55 and is critical for membrane targeting in epithelial cells

“…Considerable evidence now supports the view that the 135 kDa isoform of protein 4.1R plays a functional role in the regulation of cell division and growth, although the precise physiological basis of this function in the proliferation and differentiation pathways remains unknown [24–26]. Like other family members, the protein 4.1R is expressed in a variety of non-erythroid cells and its gene function is regulated by a complex array of alternatively spliced exons in both erythroid and non-erythroid cells [21, 23].…”

Regulated Fox-2 isoform expression mediates protein 4.1R splicing during erythroid differentiation

“…The 80-kDa red blood cell protein 4.1R is the prototype of a diverse array of 4.1R isoforms whose expression is regulated by coupled transcription and splicing, 1 use of alternative translation initiation sites, 2,3 alternative pre-mRNA splicing, 4,5 and posttranslational modification. 6,7 The expression of exon 16, which encodes peptides critical for the spectrin-actin binding, is tightly regulated during erythroid differentiation. Exon 16 is absent from the great majority of 4.1R mRNA in preerythroid cells, but predominates in late erythroid cell 4.1R mRNA.…”