Related Experiment Video

Updated: Aug 6, 2026

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
12:26

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Published on: May 3, 2018

Neuronal nuclear organization is controlled by cyclin-dependent kinase 5 phosphorylation of Ras Guanine nucleotide

Sashi Kesavapany1, Tej Kumar Pareek, Ya-Li Zheng

  • 1Cytoskeletal Protein Regulation Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.

Neuro-Signals
|August 22, 2006
PubMed

Insights

Cyclin-dependent kinase 5 (Cdk5) phosphorylates RasGRF1 at serine 731, triggering its degradation and regulating cellular organization. This Cdk5-mediated phosphorylation is crucial for maintaining normal cell structure and function.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Neuroscience

Background:

  • RasGRF1 is a Ras guanine nucleotide exchange factor (RasGEF) involved in Ras and Rac GTPase activation.
  • RasGRF1 is a phosphoprotein known to be phosphorylated by various kinases, including PKA, Ack1, and Src.
  • The precise regulation of RasGRF1 levels and its impact on cellular processes require further elucidation.

Purpose of the Study:

  • To investigate the interaction between RasGRF1 and Cdk5.
  • To determine the role of Cdk5-mediated phosphorylation of RasGRF1 in regulating its stability and cellular function.
  • To explore the consequences of altered RasGRF1 levels on nuclear organization in mammalian cells and neurons.

Main Methods:

  • Co-immunoprecipitation assays to demonstrate interaction between RasGRF1 and Cdk5.
  • In vitro kinase assays to confirm phosphorylation of RasGRF1 by Cdk5 at serine 731.
  • Western blotting to assess RasGRF1 protein levels in wild-type and Cdk5 knockout neurons, and after Cdk5 transfection.
  • Overexpression studies in mitotic cells and primary neurons to observe the effects on nuclear morphology.

Main Results:

  • RasGRF1 directly interacts with and is phosphorylated by Cdk5 on serine 731.
  • Cdk5-mediated phosphorylation of RasGRF1 on serine 731 promotes its degradation via a calpain-dependent pathway.
  • Cortical neurons from Cdk5 knockout mice exhibit elevated RasGRF1 levels, which are reduced upon wild-type Cdk5 transfection.
  • Overexpression of RasGRF1 leads to nuclear disorganization in mitotic cells, a phenotype rescued by co-expression with active Cdk5.
  • Elevated RasGRF1 levels in neurons induce nuclear condensation and fragmentation, while reduced RasGRF1 levels also result in nuclear condensation.

Conclusions:

  • Cdk5-mediated phosphorylation of RasGRF1 at serine 731 is a key regulatory mechanism controlling its protein stability.
  • The dynamic regulation of RasGRF1 levels by Cdk5 is essential for maintaining proper cellular and nuclear organization.
  • These findings highlight a novel pathway involving Cdk5 and RasGRF1 in cellular homeostasis and neuronal function.

More Related Videos

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells10:27

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Published on: March 9, 2012

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and GTPase-linked Immunosorbent Assay13:51

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and GTPase-linked Immunosorbent Assay

Published on: November 11, 2018

Related Concept Videos

Small GTPases - Ras and Rho01:24

Small GTPases - Ras and Rho

Ras and Rho are small monomeric GTPases that act downstream of receptor tyrosine kinase (RTK) and regulate various cellular processes. These GTPases switch between active and inactive states by binding to guanine nucleotides.
Three regulatory proteins control their activity:
The Ras Gene02:38

The Ras Gene

The Ras-gene-encoded proteins are regulators of signaling pathways controlling cell proliferation, differentiation, or cell survival. The Ras-gene family in humans constitutes three primary members—the HRas, NRas, and KRas. These genes code for four functionally distinct yet closely related proteins—the HRas, NRas, KRas4A, and KRas4B. The involvement of mutant Ras genes in human cancer was first discovered in 1982 and is among the most common causes of human tumorigenesis.
Ras is a superfamily...
Directionality of Nuclear Transport01:42

Directionality of Nuclear Transport

Ras-related nuclear protein or Ran is a small G protein that cycles between its GTP and GDP bound states. Ran specific regulators, a Ran GTPase Activating Protein or RanGAP present in the cytosol and a Ran guanine nucleotide exchange factor or RanGEF present inside the nucleus regulate GTP/GDP exchange. A high concentration of GTP inside the cells, in addition to this asymmetric distribution of  Ran-specific regulators, leads to a higher RanGTP concentration inside the nucleus. This...
Regulation of Nuclear Protein Sorting01:45

Regulation of Nuclear Protein Sorting

Nuclear protein sorting regulates nucleus composition and gene expression, crucial for determining the fate of a eukaryotic cell. Hence, the entry and exit of molecules across the nuclear envelope is a tightly controlled process. Nuclear protein sorting can be inhibited by one of the following ways: 1) masking cargo signal sequences, 2) modifying the nuclear receptor's affinity for cargo, 3) controlling the nuclear pore size, 4) retaining the cargo during its transit to the cytosol or the...
MAPK Signaling Cascades01:07

MAPK Signaling Cascades

Mitogen-activated protein kinase, or MAPK pathway, activates three sequential kinases to regulate cellular responses such as proliferation, differentiation, survival, and apoptosis. The canonical MAPK pathway starts with a mitogen or growth factor binding to an RTK. The activated RTKs stimulate Ras, which recruits Raf or MAP3 Kinase (MAPKKK), the first kinase of the MAPK signaling cascade. Raf further phosphorylates and activates MEK or MAP2 Kinases (MAPKK), which in turn phosphorylates MAP...
GTPases and their Regulation02:14

GTPases and their Regulation

Guanine nucleotide-binding proteins (G-proteins), also known as GTPases, are a superfamily of proteins that regulate many cellular processes, such as cell signaling, vesicular transport, and the regulation of cell shape and motility. Mutation or dysfunction of these proteins can lead to disease. There are around 40,000 known G-proteins that can broadly be classified into two groups ‒  small G-proteins consisting of a single domain and large multi-domain G-proteins.
Large G-proteins, also known...