Erythrocyte protein 4.1 (P4.1) is an 80 kD protein that stabilizes the spectrin-actin junction of the spectrin cortical skeleton. It is one member of a complex family of alternatively spiced gene products that arise from the 4.1R gene. In non-erythroid cells, an alternative upstream initiation site typically generates a larger form of the protein, 135 kD, with an extended amino-terminus. Antibodies to this amino-terminal extension react with cell nuclei, centrosomes, and parts of the mitotic apparatus in dividing cells. Work now submitted for publication reveals that at least one of the 135 kD transcripts of the 4.1R gene specifically interacts with the nuclear mitotic apparatus protein (NuMA), that they co-localize in the nucleus during interphase, and redistribute together to the spindle poles early in mitosis. This form is also specifically phosphorylated during mitosis in HeLa and C2C12 cells by p34cdc2 kinase in vitro, and undergoes cyclic phosphorylation in vivo. Microinjection of antibodies to P4.1135 results in micronuclei and multiple astral arrays. These findings have extended our understanding of P4.1, establish that at least some 4.1 isoforms are nuclear associated, and suggest that P4.1 plays a pivotal role in nuclear architecture and spindle formation during mitosis. In the proposed studies, this hypothesis will be further explored and the mechanisms of its effects determined by immunodepletion with epitope specific antibodies in both cell-free reconstitution systems for mitotic asters and in cultured cells, and by using dominant negative mutants overexpressed in cultured cells, with rescue by the wild types. The relevance of phosphorylation will be evaluated by analyzing the behavior of mutants altered at the conserved cdc2 phosphorylation site (thr-60). It is anticipated that these studies will reveal the way that this putative cytoskeletal protein functions in cell division.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29HL061295-02
Application #
6139295
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1999-01-01
Project End
2002-12-31
Budget Start
2000-01-01
Budget End
2000-12-31
Support Year
2
Fiscal Year
2000
Total Cost
$113,400
Indirect Cost
Name
Johns Hopkins University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Huang, Shu-Ching; Zhou, Anyu; Nguyen, Dan T et al. (2016) Protein 4.1R Influences Myogenin Protein Stability and Skeletal Muscle Differentiation. J Biol Chem 291:25591-25607