Related Experiment Video

Updated: Aug 26, 2026

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle
10:05

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Published on: March 5, 2019

SPAK kinase is a substrate and target of PKCtheta in T-cell receptor-induced AP-1 activation pathway

Yingqiu Li1, Junru Hu, Randi Vita

  • 1Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121, USA.

The EMBO Journal
|February 28, 2004
PubMed

Insights

We identified SPAK, a mitogen-activated protein kinase (MAPK), as a key activator of Protein kinase C-theta (PKCtheta) signaling in T cells. This interaction is crucial for T-cell activation and AP-1 stimulation.

Area of Science:

  • Immunology
  • Cell Signaling
  • Molecular Biology

Background:

  • Protein kinase C-theta (PKCtheta) is essential for T-cell activation, regulating transcription factors AP-1 and NF-kappaB.
  • Understanding the upstream regulators of PKCtheta is critical for deciphering T-cell signaling pathways.

Purpose of the Study:

  • To identify and characterize novel kinases that interact with and regulate PKCtheta.
  • To elucidate the role of SPAK in PKCtheta-mediated T-cell activation.

Main Methods:

  • Co-immunoprecipitation to identify interacting proteins.
  • Kinase assays to measure catalytic activity.
  • Site-directed mutagenesis to probe functional regions.
  • RNA interference (RNAi) and dominant-negative mutants to assess functional roles.
  • Western blotting to detect protein phosphorylation and activation.

Main Results:

  • SPAK, a Ste20-related MAPK, was identified as a specific interactor of PKCtheta, binding to its C-terminus.
  • T-cell receptor/CD28 costimulation enhanced SPAK-PKCtheta interaction and SPAK catalytic activity.
  • PKCtheta directly phosphorylated SPAK at Ser-311, a critical event for SPAK activation.
  • SPAK activation and subsequent AP-1 activation were significantly impaired in PKCtheta-deficient T cells.
  • SPAK and PKCtheta synergistically activated AP-1, dependent on the SPAK-PKCtheta interaction region and Ser-311 phosphorylation.

Conclusions:

  • SPAK is a novel substrate and direct target of PKCtheta in T-cell signaling.
  • The SPAK-PKCtheta interaction is essential for TCR/CD28-induced T-cell activation, specifically promoting AP-1 transcription factor activity.
  • This study defines a new signaling axis (PKCtheta-SPAK) crucial for adaptive immune responses.

More Related Videos

Assaying Protein Kinase Activity with Radiolabeled ATP08:05

Assaying Protein Kinase Activity with Radiolabeled ATP

Published on: May 26, 2017

Understanding the Development of Compensatory Pathways in a Mutant Malaria Parasite Harbouring Hypomorphic Allele of Plant-Like Kinases09:13

Understanding the Development of Compensatory Pathways in a Mutant Malaria Parasite Harbouring Hypomorphic Allele of Plant-Like Kinases

Published on: November 22, 2024

Related Concept Videos

The JAK-STAT Signaling Pathway01:20

The JAK-STAT Signaling Pathway

Several cytokine receptors have tightly bound Janus kinase or JAK proteins attached at their cytosolic tail. Small signaling molecules such as cytokines, growth hormones, or prolactins bind to the cytokine receptors and initiate their dimerization. The dimerization brings the cytosolic JAKs together that trans-phosphorylate and activates each other. The activated JAKs now phosphorylate cytosolic tails of the cytokine receptors, which serve as binding sites for adaptor proteins such as  SH2...
cAMP-dependent Protein Kinase Pathways01:25

cAMP-dependent Protein Kinase Pathways

Cyclic Adenosine Monophosphate (cAMP) is an essential second messenger that activates protein kinase A (PKA) and regulates various biological processes. A single epinephrine molecule binds to GPCR and activates several heterotrimeric G proteins, each stimulating multiple adenylyl cyclase, amplifying the signal, and synthesizing large numbers of cAMP molecules. Small changes in cAMP concentration affect PKA activity. The binding of four cAMP molecules induces a conformational change in PKA,...
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...
PI3K/mTOR/AKT Signaling Pathway01:22

PI3K/mTOR/AKT Signaling Pathway

The mammalian target of rapamycin  (mTOR) is a serine/threonine kinase that regulates growth, proliferation, and cell survival in response to hormones, growth factors, or nutrient availability. This kinase exists in two structurally and functionally distinct forms: mTOR complex 1  (mTORC1) and mTOR complex 2  (mTORC2). The first form (mTORC1) is composed of a rapamycin-sensitive Raptor and proline-rich Akt substrate, PRAS40. In contrast,  mTORC2 consists of a rapamycin-insensitive companion...
Amplifying Signals via Enzymatic Cascade01:22

Amplifying Signals via Enzymatic Cascade

When a ligand binds to a cell-surface receptor, the receptor's intracellular domain changes shape, which may either activate its enzyme function or allow its binding to other molecules. The initial signal is amplified by most signal transduction pathways. This means that a single ligand molecule can activate multiple molecules of a downstream target. Proteins that relay a signal are most commonly phosphorylated at one or more sites, activating or inactivating the protein. Kinases catalyze the...
Interactions Between Signaling Pathways01:19

Interactions Between Signaling Pathways

Signaling cascades usually lack linearity. Multiple pathways interact and regulate one another, allowing cells to integrate and respond to diverse environmental stimuli.
Convergence and divergence, and cross-talk between signaling pathways
Two distinct signaling pathways can converge on a single functional unit, which may either be a single protein or a complex of proteins. The response is either functionally distinct or synergistic between the two pathways but different from the response...