Phosphorylation of a New Brain-specific Septin, G-septin, by cGMP-dependent Protein Kinase

doi:10.1074/jbc.275.14.10047

Phosphorylation of a New Brain-specific Septin, G-septin, by cGMP-dependent Protein Kinase*

From the ^‡Cell Signalling Unit, Children's Medical Research Institute, Wentworthville 2145, New South Wales, Australia, the ^§Department of Medical Biochemistry, Faculty of Medicine and Health Sciences, University of Newcastle, Newcastle 2308, New South Wales, Australia, the ^¶Department of Molecular Oncology, Graduate School of Medicine, Kyoto University, Kyoto 606, Japan, the ^‖John Curtin School of Medical Research, Australian National University, Canberra 0200, Australian Capital Territory, Australia, and the ^**Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Szeged H-6701, Hungary

Abstract

The septins are a family of GTPase enzymes, some of which are required for the cytokinesis stage of cell division and others of which are associated with exocytosis. We purified and cloned the cDNA for a 40-kDa protein from rat brain that is a substrate for type I cGMP-dependent protein kinase (PKG). The amino acid sequences of two tryptic peptides of P40 showed high homology to the septins. Molecular cloning revealed the 358-amino acid P40 to be a new member of the septin family. P40 was named G-septin, as it is phosphorylated in vitro by PKG, but relatively poorly by the related cAMP-dependent protein kinase and not by protein kinase C. Two splice variants of G-septin (α and β) were found with distinct N and C termini, but a common GTPase domain. G-septin lacks the C-terminal coiled-coil domain characteristic of all other mammalian septins and uniquely has two predicted phosphorylation site motifs for type I PKG. Photoaffinity labeling with [α-³²P]GTP confirmed that G-septin is a GTP-binding protein. Northern blotting showed that G-septin mRNA (5.0 kilobases) is highly expressed in brain and undetectable in 12 other tissues, indicating that the G-septins are primarily neuronal proteins. Very low levels of 6.0-, 3.4-, and 2.6-kilobase transcripts were found in testis. Our results reveal a new class of brain-specific septins that may be regulated by PKG in neurons.

Footnotes

↵* This work was supported by grants from the Australian National Health and Medical Research Council and the National Heart Foundation, a University of Newcastle postgraduate research scholarship (to J. X.), and an International postgraduate research scholarship (to J. X.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) and .
↵‡ To whom correspondence should be addressed: Cell Signalling Unit, Children's Medical Research Inst., Locked Bag 23, Wentworthville 2145, NSW, Australia. Tel: 61-2-96872800; Fax: 61-2-96872120; E-mail: phrobins@mail.usyd.edu.au.
↵2 J. Xue, X. Wang, C. S. Malladi, M. Kinoshita, P. J. Milburn, I. Lengyel, J. A. P. Rostas, and P. J. Robinson, unpublished data.
Abbreviations:

GTPγS

guanosine 5′-O-(3-thiotriphosphate)

PKG

cGMP-dependent protein kinase

PKA

cAMP-dependent protein kinase

PKC

protein kinase C

PKI

PKA inhibitor

HPLC

high performance liquid chromatography

PAGE

polyacrylamide gel electrophoresis

PCR

polymerase chain reaction

bp

base pair(s)

kb

kilobase(s)

pCPT-

p-chlorophenylthio-
- Received July 16, 1999.
- Revision received January 18, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.