Differential Requirement for RhoA GTPase Depending on the Cellular Localization of Protein Kinase D

Michael J Mullin,Kurt Lightfoot,Ulrica Marklund,Doreen A Cantrell

doi:10.1074/jbc.m603591200

Abstract

This study explores the links between the GTPase RhoA and the serine kinase protein kinase D (PKD) during thymocyte development. The rationale is that RhoA and PKD regulate common biological responses during T cell development, but there is nothing known about their interdependence. In fibroblasts, Rho function is required for activation of PKD catalytic activity. However, the data show that activation of Rho is neither sufficient nor essential for PKD activation in T cells. One alternative explanation for the apparent convergence of PKD and Rho signaling in T cells is that PKD responses might be Rho-dependent. To address this latter possibility, we probed the Rho requirements for the actions of constitutively active PKD mutants in pre-T cells of transgenic mice. Active PKD can localize to either the plasma membrane or the cytosol, and we therefore compared the Rho requirements for the actions of membrane- or cytosol-localized PKD. Here we show that membrane-localized PKD regulation of pre-T cell differentiation is Rho-dependent, but the actions of cytosol-localized PKD are not. These studies demonstrate that a Rho requirement for PKD activation is not ubiquitous. Moreover, links between PKD and Rho are determined by the cellular location of PKD.

Highlights

Protein kinase D1 is a member of a conserved family of serine/threonine kinases that includes PKD2 and protein kinase C (PKC)␯/PKD3 [1, 2]
protein kinase D (PKD) Activation Does Not Require Endogenous RhoA Function—The activity of RhoA is required for PKD activation in fibroblasts and intestinal cells [19] [17, 20]
PKD activation is mediated by phosphorylation of residues Ser-744 and Ser-748 in the catalytic domain of the enzyme, and this results in immediate autophosphorylation of PKD1 on a Cterminal autophosphorylation site, Ser-916

Summary

EXPERIMENTAL PROCEDURES

CDNA Constructs—Vectors encoding a chimeric fusion protein between green fluorescent protein (GFP) and wild type PKD1 or the constitutively active membrane-targeted PKD have been described before [6, 12, 26]. pEF-link vectors expressing N-terminal 9E10 epitope-tagged C3 transferase or V14RhoA have been described previously [27]. Stimulated cells were washed in ice-cold phosphate-buffered saline and subjected to Western blot analysis. Jurkat cells were transfected as described above with an SRE-CAT reporter gene comprising V14RhoA (2 ␮g) alone or with C3 transferase constructs (5 ␮g). Transgenic mice expressing the constitutively active membrane-targeted PKD or cytosolic targeted PKD in the thymus under the control of CD2 promoter and locus control region have been described previously [12]. Intracellular TCR␤ staining was carried out on thymocyte subpopulations by antibody staining, which were subsequently fixed in 1% paraformaldehyde for 10 min at room temperature, washed in PBS, and permeabilized with saponin buffer Permeabilized cells were incubated with PEconjugated TCR␤ antibody for 45 min at room temperature in saponin buffer, washed in saponin buffer, and analyzed by FACS. Cell surface binding sites were blocked using biotinylated TCR␤ antibody, and specificity of staining was verified by parallel staining with PE-conjugated isotypematched control antibody (Armenian hamster IgG group 2␭)

RESULTS

Responses Induced by PKD in T

DISCUSSION